Decoding the proregenerative competence of regulatory T cells through complex tissue regeneration in zebrafish

Abstract

Regulatory T cells (T_regs) are specific subtype of T cells that play a central role in sustaining self‐antigen tolerance and restricting inflammatory tissue damage. More recently, additional direct functions of T_regs in mammalian tissue repair have emerged, but the regenerative potential of T_regs in non‐mammalian vertebrates has not been explored despite the latter possessing a highly developed adaptive immune system. Why complex organs such as the caudal fin, heart, brain, spinal cord and retina regenerate in certain non‐mammalian vertebrates, but not in mammals, is an interesting but unresolved question in the field of regenerative biology. Inflammation has traditionally been thought to be an impediment to regeneration due to the formation of scars. Regenerative decline in higher organisms has been speculated to be the evolutionary advent of adaptive immunity. Recent studies, however, have shown that the innate inflammatory response in non‐mammalian organisms is required for organ regeneration. It has also been found that highly advanced adaptive immunity is no longer incompatible with regeneration and for that, T_regs are important. Zebrafish regulatory T cells (zT_regs) migrate rapidly to the injury site in damaged organs, where they facilitate the proliferation of regeneration precursor cells by generating tissue‐specific regenerative factors by a process distinct from the canonical anti‐inflammatory pathway. We review both reparative and proregenerative roles of T_regs in mammals and zebrafish, respectively, and also give an overview of the forkhead box protein 3 (FoxP3) ‐dependent immunosuppressive function of T_regs in zebrafish, which makes it a useful model organism for future T_reg biology and research.

In mammals, T_{reg s} modulate inflammation and lead to tissue repair through inhibition of neutrophil and inflammatory macrophage, and exerting immunosupressive role by releasing anti‐inflammatory cytokines. In addition to immunosuppressive role, zT_{reg s} secerete proregenerative factors in injury niche for successful tissue regeneration. zT_{reg s} display unique organ‐specific proregenerative factor secretion ability to induce tissue resident progenitor cell proliferation during regeneration.

INTRODUCTION

Regulatory T cells (T_regs) represent around 5%–10% of the T cell population in peripheral circulation and are important mediators of peripheral self‐ and non‐self antigenic tolerance [1]. This immunoregulatory control of T_regs is achieved by direct cell‐to‐cell interaction via the production of anti‐inflammatory cytokines that inhibit innate immune cells, the functioning of antigen‐presenting cells (APCs) as well as the functioning of the B cells, CD4⁺ or CD8⁺ effector T (T_eff) cells [2]. Although T_regs are not the only immune cells responsible for immune suppression, they are considered to play the most important role in case of immunosuppression.

T_regs belong to a subset of CD4⁺ T cells, are of two main types – natural T_reg and adaptive (or induced) T_reg. Natural T_regs characteristically express some cell surface markers, which include interleukin (IL)‐2 receptor alpha chain (CD25) and cytotoxic T lymphocyte antigen‐4 (CTLA‐4). Forkhead box P3 (FoxP3) transcription factor is another marker that is expressed in the nucleus of natural T_regs. They secrete IL‐10 and transforming growth factor (TGF)‐β, preventing effector T cells (T_eff) from proliferation. They suppress the release of proinflammatory cytokines such as interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α in response to mitogenic and antigenic stimulation. Conversely, adaptive T_regs are FoxP3⁺ antigen‐specific immunosuppressors that are generated from CD4⁺ T cells stimulated with TGF‐β and IL‐10, and act identically to normal T_regs [3, 4, 5, 6]. FoxP3 expression is critical for T_regs to act as immunosuppressive cells [7, 8]. There are several signaling cascades that are responsible for T_reg production in addition to FoxP3, which is the master regulator of T_reg. TGF‐β‐SMAD signaling also plays an essential role in developing T_reg from naive CD4⁺ T cells [9].

FoxP3 regulation of T_reg is crucial for the resolution of inflammation. In humans, a FoxP3 gene mutation that obstructs the FoxP3 function leads to a rare human disease – immunodysregulation polyendocrinopathy enteropathy X‐linked (IPEX) – in which persistent inflammatory conditions including increased cytokine synthesis, are among the main symptoms [6, 10].

T_regs are known to suppress a variety of immune cells, including B cells, natural killer (NK) cells, natural killer T (NKT) cells and often CD4⁺ or CD8⁺ T cells. The suppression of conventional CD4⁺ T cells involves a number of mechanisms. Depending on the site of suppression, the activation states of the target cell and the T_reg itself, different pathways exist and participate [11]. T_regs rapidly suppress T cell receptor (TCR)‐induced signaling pathways such as Ca²⁺, nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NF‐κB) signaling in conventional T cells (T_conv). In order to perform their suppressive role on target cells, they also produce immunosuppressive molecules such as adenosine and other immunosuppressive cytokines (IL‐10, TGF‐β, IL‐35) [11, 12]. Human T_regs have been found to express the serine protease granzyme‐A, which induces the destruction of effector cells through a perforin‐dependent pathway [13]. T_regs also suppress T cells partially by down‐regulating co‐stimulatory molecules such as CD80 and CD86 on APCs via CTLA‐4 [8, 12, 14].

REGULATORY T CELLS IN TISSUE REPAIR AND REGENERATION

T_regs have also been linked to reducing tissue damage in addition to securing immunological tolerance to chronic exposures of ‘self’ and ‘non‐self’ antigens [9, 15]. Tissue repair may be aided by T_regs in addition to the reduction of tissue damage by suppressing inflammatory response after infection [15]. T_reg‐mediated tissue repair is induced by suppressing the production of proinflammatory chemokines, endothelial cell activation, and proinflammatory responses of innate and adaptive immune cells [16]. T_regs are found not only in secondary lymphoid organs but also in other non‐lymphoid bodies that quickly recruit circulatory T_regs , while resident T_regs expand in response to tissue damage or injury [17]. Therefore, through the development of mediators on parenchymal cells, T_regs probably play a direct role in tissue repair.

Infiltrating T_regs influence tissue‐resident stem cell proliferation and differentiation following damage in several mouse tissues (e.g. skeletal muscle, lung epithelium and skin). In a dystrophic mouse model, T_reg depletion during the repair phase increased the proinflammatory infiltrate and hindered muscle repair, while therapies that increased or decreased T_reg activities reduced or improved muscle damage, respectively [18]. According to researchers, muscle T_regs secrete the growth factor Amphiregulin, which directly affects muscle satellite cells in vitro and enhances in vivo muscle repair [18]. Another study found that after cardiotoxin (CTX) injury in mice, a specific CD4⁺CD25⁺FoxP3⁺ population of T cells have been engaged in the repair process of skeletal muscles, which regulates the functioning of skeletal muscle precursor cells in addition to their well‐established role as immune system regulators [19]. In another study, Arpaia et al. demonstrated that T_regs may directly exert tissue repair ability, at least to some extent, through the synthesis of amphiregulin utilizing influenza lung infection in mice as a model of pathogen‐induced inflammation and tissue damage [15]. In the skin of adult mice, a subgroup of T_reg releases the epidermal growth factor receptor (EGFR) to promote wound healing [20]. Another research study of transcriptional and phenotypical profiling of T_regs and hair follicular stem cells conducted by Ali et al. supported this repairing role of T_reg, which showed that skin‐resident T_regs exclusively express Jagged 1, a member of the Notch family of ligands which, in turn, promotes the activity of hair follicle‐stimulating cells (HFSCs) [21]. Recently, mature T lymphocytes with T_reg‐like attributes have been recognized using the FoxP3 orthologue as a marker in zebrafish, and these cells have been found to play a key role in immunoregulation [22]. These findings show that T_regs have a distinct protective role in tissue damage, repair and maintenance, but this function is triggered in response to signals that are distinct from those that activate their immune‐suppressing capability.

REGULATORY T CELLS AS AN INFLAMMATORY MODULATOR IN TISSUE INJURY NICHE

T_regs have also shown that they can regulate both innate as well as adaptive immune systems to facilitate tissue repair by inflammatory modulation. A cascade of immune processes is activated before a new tissue forms post‐injury and T_regs are involved in all these different steps. T_regs can impede neutrophil extravasation through IL‐10 and thus neutralize inflammatory cytokine secretion (e.g. IL‐6, IFN‐γ, TNF‐α and IL‐1β) at the beginning of inflammation [23]. Further, T_regs are capable of facilitating neutrophil apoptosis and induce macrophages for the phagocytic clearance of dead neutrophils [17]. T_regs also release several anti‐inflammatory cytokines (e.g. IL‐4, IL‐10, IL‐13), which impair monocyte activity and survival while also stimulating the polarization of macrophages into an anti‐inflammatory phenotype (M2) [24]. In addition, T_regs naturally restrict CD4⁺ and CD8⁺ T cell‐mediated inflammation through the production of IL‐10, TGF‐β and IL‐35 [25, 26]. As a whole, these T_reg‐mediated processes lead to tissue repair through the inhibition of neutrophils and inflammatory macrophages and also by CD4⁺ and CD8⁺ T cell involvement.

In skeletal muscle repair, IL‐33 assists in recruiting T_regs to the injured site which inhibits inflammation caused by M1 macrophages, promoting muscle repair [27]. In the case of cardiac injury, activated CD4⁺CD25⁺ T cell infiltration has been found within the infarcted and distant regions of the myocardium in patients with myocardial infarction (MI) [28]. Through CD39 expression, which promotes extracellular nucleotide degradation to form adenosine, in vitro‐activated T_regs reduce myocardial damage [29]. Therefore, in the case of cardiac injury, T_regs can act through CD39‐mediated adenosine formation [29]. T_regs also enhance post‐MI healing by modulating monocytes and macrophages. Following MI, decreased cardiac activity due to enhanced M1 macrophage invasion and marked left ventricular dilation were observed in cases of T_reg depletion in FoxP3^DTR mice or anti‐CD25 monoclonal antibody (mAb) treatment [30]. Conversely, superagonistic anti‐CD28 mAb application preferentially activates T_reg, leading to higher T_reg invasion into the infracted myocardium after MI, induces macrophages in the repairing myocardium to adopt the M2 phenotype and also reduces ventricular ruptures, resulting in improved survival [30]. According to another report, CCR5 knock‐out mice exhibit disrupted T_reg infiltration as well as inappropriate remodeling and cardiac dysfunction after MI [31]. Therefore, inflammation, extensive matrix depletion and adverse remodeling after MI are suppressed as a result of CCR5‐mediated T_reg recruitment [31].

T_regs suppress the inflammatory activity of M1 macrophage in the lungs and promote the differentiation of impaired type 2 alveolar epithelial cells (AECII) into type 1 alveolar epithelial cells (AECI), and this step is mediated by binding of CD103 ligand to E‐cadherin receptor [32]. T_regs could also stimulate the differentiation of progenitor bronchioalveolar stem cells (BASC) into AECII cells [33]. At the same time, T_regs act via C‐X‐C motif chemokine ligand 12 (CXCL12) to prevent the recruitment proliferation of fibrocytes and prevent fibrosis [33]. T_regs are likely to be recruited in bone repair through C‐C motif chemokine ligand 22 (CCL22), which is used to induce the differentiation of the osteoblast progenitors by inhibiting T helper type 1 (Th1) and M1 macrophages [34].

In the central nervous system (CNS), IL‐33 recruits T_regs to perform a reparative function by promoting M2 macrophage polarization to aid remyelination and oligodendrocyte differentiation [35]. Also, CCN3 promotes myelination and the differentiation of oligodendrocyte progenitor cells in vitro and ex vivo [36]. According to another study, while mouse T_regs invade the CNS minimally they are insufficient to encourage regeneration despite having a strong immunomodulatory impact [37].

ZEBRAFISH REGULATORY T CELLS (zT_regs) AND THEIR ROLE IN COMPLEX TISSUE REGENERATION

Unlike mammals, the zebrafish genome has two FOXP3 orthologs, foxp3a and foxp3b, which share the same functional domains as mammalian FoxP3 and may serve a redundant role in T_reg formation and its activity [22, 38, 39]. However, although foxp3a embryonic expression rises as T cells mature, foxp3b expression remains substantially constant throughout development in zebrafish [22], suggesting that zebrafish foxp3a is the most probable homolog of mammalian FOXP3. An interesting study [38] first described the Foxp3 function in zebrafish when they were observed through a series of biochemical investigations showing that zebrafish Foxp3a (termed ‘zFoxp3’), like mammalian FOXP3, may have an immunomodulatory role in zebrafish and that FOXP3 involvement in immunosuppressive mechanisms is evolutionarily conserved in non‐mammalian vertebrates. The in vivo function of zebrafish foxp3a has also confirmed an immunomodulatory role in homeostasis by creating a zebrafish mutant with loss‐of‐function foxp3a alleles by using transcription activator‐like effector nucleases (TALEN) [22, 39]. Further, It has been discovered that foxp3a, a zebrafish ortholog of the mammalian FOXP3 gene, mainly defines a fraction of T cells with the conserved gene expression profile of mammalian T_regs [40].

Following tissue injury, the regeneration competent animals require the proliferation of tissue‐resident precursor cells for successful regeneration. Zebrafish regenerative biology is an open area of thorough investigation and the credits for injury‐induced regeneration are attributed to the neural stem cells (ependymo‐radial glial cells) in the spinal cord [41, 42], cardiomyocytes in the heart [43, 44] and Müller glia in the retina [45, 46]. Zebrafish are armoured with both an innate and adaptive immune system akin to mammals and exhibit nearly all types of immune cells [47]. Components of the innate immune system play a crucial role in injury‐induced regenerative responses in the non‐mammalian system and have been thoroughly investigated in multiple organ systems such as brain, limb, caudal fin, heart and retina [48]. Later findings not only challenged the popular dogma that adaptive immunity is incompatible with regeneration, but also confirmed the pivotal roles played by one species of T lymphocyte towards a proregenerative direction. The advent of zT_regs in the context of zebrafish regeneration has marked a major paradigm shift in our understanding of existing regenerative immunology.

T_regs are known to exert immunosuppressive roles, but recent studies reveal their regenerative potential in the zebrafish model system. Following a transection injury [42, 49] in zT_reg cell‐depleted spinal cords, the rostral and caudal stumps remain disconnected and the locomotor activity also fails to become restored up to a time‐point by which zebrafish can regain their pre‐injury swimming performance. Additionally, clear evidence in favor of the co‐presence of Foxp3a‐expressing zT_reg and SRY‐box transcription factor 2 (Sox2) and proliferating cell nuclear antigen (PCNA)‐expressing proliferating neural progenitor cells was found. Post‐injury depletion of zT_reg leads to the reduction of Sox2⁺ neural progenitors at 7 days post injury (dpi) spinal cord and HuC/D⁺ newly born neurons [40]. Taken together, these findings show us the pivotal role played by zT_reg in the proliferation of neural progenitor cells by secreting neurogenic factors such as neurotrophin‐3 (Ntf3), glial cell‐derived neurotrophic factor‐a (Gdnfa) and nerve growth factor beta (Ngfb) in the injury‐induced spinal cord, and thus facilitate spinal cord regeneration in zebrafish [40].

The emergence of proregenerative roles of zT_reg has expanded our knowledge of retinal regeneration [45, 46, 50, 51, 52, 53, 54, 55]. Following a poke injury in zT_reg cell‐depleted zebrafish retina, the layered structure remains highly disorganized [40]. The role of zT_reg in the proliferation of precursor cells has been further confirmed by the presence of foxp3a expressing zT_reg in the vicinity of PCNA⁺ proliferating Müller glia [56]. Depletion of zT_reg after a retinal injury leads to a significant reduction in the Müller glia proliferation in the damaged retina [40]. zT_regs also produce several growth factors in the damaged retina, which include insulin‐like growth factor 1 (Igf1), heparin‐binding EGF‐like growth factor a (Hb‐egfa), epidermal growth factor (Egf) and platelet‐derived growth factor alpha polypeptide a (Pdgfaa). In the injury‐induced retina the up‐regulation of Igf1 is marked significantly, making it the most important zT_reg‐derived Müller glia‐inducing growth factor [40].

Similar to the spinal cord and retinal regeneration, cardiac regeneration [43, 44, 57, 58] in zebrafish is also aided by its T_regs. In regenerating hearts, zebrafish T_regs are seen near or in direct contact with the proliferating cardiomyocytes, indicating a paracrine role in myocardial regeneration. It is evident from some findings that, following cardiac damage, zT_regs infiltrate the regenerating myocardium from the bloodstream, where mature T_regs are activated and expanded in number [59, 60, 61, 62]. Myocyte enhancer factor‐2 (Mef2) is one of the few signature cardiac transcription factors and the MEF2⁺ proliferating cardiomyocytes decrease in T_reg‐depleted injury‐induced zebrafish heart, indicating a clear role of zT_reg in cardiac regeneration [40, 63]. As a repercussion to cardiac injury, several cardiomyocyte mitogens namely the neuregulin 1 (Nrg1), insulin‐like growth factor 2a (Igf2a), insulin‐like growth factor 2b (Igf2b), and platelet‐derived growth factor subunit B (Pdgfb) are secreted from zT_reg. Among these mitogens, a conspicuous up‐regulation of n rg1 is observed in the regenerating zebrafish heart. NRG1 signalling is reported to induce cardiomyocyte proliferation in both zebrafish and mammals, and the administration of human NRG1 can rescue cardiomyocyte proliferation in the injury‐induced T_reg‐depleted heart of zebrafish [40].

Moreover, zT_reg has been proved to be effective beyond its immunosuppressive role to facilitate regeneration in an organ‐specific manner. These cells are largely absent in uninjured organs, but rapidly migrate to the site of injury during 3–7 days post‐injury and promote the proliferation of regeneration precursor cells in the injured organs by producing tissue‐specific regenerative factors, such as Ntf3, for the spinal cord, and include Nrg1 for the heart and Igf1 for the retina [40] (Figure 1). Similar to the regeneration competent neonate mice, amphiregulin (Areg) is also another factor produced by zT_reg, but in a non‐tissue‐specific manner [15, 18, 59]. It has also been found that the proregenerative role played by the zT_reg is independent of its classical immunosuppressive function [64]. Improved zT_reg recruitment has been observed in zebrafish following tail amputation after treating it with a dopamine agonist and this indicates the importance of dopaminergic signaling in the regulation of zT_{re g} recruitment [65]. In the following sections, we discuss the tissue‐specific roles for zT_regs in organ regeneration that can open a new vista of T_reg cell‐mediated regenerative therapies in humans.

FIGURE 1.

Injury‐induced proregenerative responses of zebrafish regulatory T cells (zT_regs) in various organs. (A) Spinal cord – recruitment of zT_regs after spinal cord injury and secretion of neurotrophin‐3 (Ntf3), which facilitates proliferation of neural progenitors for successful spinal cord regeneration. (B) Retina – recruitment of zT_regs after retinal injury and secretion of insulin‐like growth factor 1 (Igf1), which facilitates proliferation of Müller glia for successful retinal regeneration. (C) Heart – recruitment of zT_regs after cardiac injury and secretion of neuregulin 1 (Nrg1), which facilitates the proliferation of cardiomyocytes for successful cardiac regeneration.

ZEBRAFISH AS A MODEL FOR REGULATORY T CELL BIOLOGY: WHAT WE DON’T KNOW

Evidence suggests that zT_regs perform an evolutionary and universal involvement in maintaining homeostasis and regeneration of different tissues by generating growth factors that selectively boost the regeneration of tissue‐specific cell types in zebrafish. By controlling stem cell proliferation and differentiation in some adult tissues, mouse T_regs also can affect regeneration. Despite having a powerful immunomodulatory impact, T_regs are insufficient to induce regeneration in poor regenerative tissues such as the heart and central nervous system in mammals [30, 37, 66]. Unexpectedly, zebrafish deficient in T_regs or FoxP3 function do not acquire the same fatal autoimmunity condition as mice lacking the T_reg function [40]. Possible redundancy of FoxP3b, which is not typically expressed in zT_regs, might explain this phenotypical difference [39]. The mild inflammatory response reported in T_reg‐deficient zebrafish may indicate that immunological responses are slowed in aquatic species [67]. Mammalian T_regs may have lost or have restricted proregenerative potential at the expense of acquiring a powerful immunosuppressive ability to harness a quick and strong inflammatory response that has developed in terrestrial animals to heal wounds [68]. Currently a number of transgenic lines of zebrafish are available, and these have further dissected out the T_reg cell functions. In the damaged spinal cord of Foxp3a:green fluorescent protein (GFP) reporter zebrafish, GFP⁺ cells were found near proliferating neural progenitor cells during spinal cord regeneration [40]. Similar results were found in cardiac regeneration, further signifying the role of zT_reg in organ regeneration in zebrafish which can only be accomplished when inflammation is restrained.

The inflammatory immune response is maintained by a variety of chemical mediators such as components of complements, vasoactive compounds, lipid mediators, cytokines, chemokines and proteolytic enzymes derived from plasma, immune cells or injured tissue niche. The same machinery has also been significantly developed in zebrafish, with the discovery of similar inflammatory mediators and receptors causing inflammatory responses by inflammasome activation. With the advent of zT_reg, zebrafish is believed to be a future model for a clearer understanding of the complex mechanisms implicated in acute and chronic inflammatory diseases [69, 70]. The use of polyclonal T_regs can potentially suppress protective immunity against tumors and infectious diseases by transferring a large number of T_regs of broad undefined specificity. Thus, developing antigen‐specific T_reg therapy will probably provide a more effective and safer alternative. Thus, T_reg therapy is in recent focus to treat autoimmune diseases – including type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease and graft‐versus‐host disease that often occur after bone marrow transplantation, organ transplant rejection and potentially to treat non‐immune diseases, such as heart disease and type 2 diabetes, Alzheimer’s disease and Parkinson’s disease [71]. According to this perspective, zT_regs would be a new tool to move forward with the advancement of future T_reg therapies.

zT_regs are critical for complex tissue regeneration and have a remarkable ability to release tissue‐specific factors tailored to the tissue‐specific progenitors [40]. An interesting recent in vivo screening study identified novel compounds that potentially recruit zT_reg to the injury niche and are implicated in tissue regeneration [65]. Investigating the zT_reg population with proregenerative tissue‐specific growth factor secretory capacity using the single‐cell sequencing method will be a future tool to develop advanced T_reg‐specific therapies for complex tissue regeneration and diseases. Thus, harnessing the T_reg numbers and functions in injured or lost tissues by enhancing and/or promoting their proregenerative function in an injured niche may be a significant step in decoding the regenerative potentials in complex tissues of humans which lack regenerative potency.

DISCUSSION AND FUTURE PERSPECTIVES

Zebrafish have largely been used as a model for studying developmental processes. It has now emerged as an extremely important system for modeling human diseases. It is a smart system similar to those of mammalian models. Moreover, the whole‐genome duplication event and subfunction specialization of gene duplicates lead to a more intricate relationship among the components implicated within the response and, as a consequence, within the inflammatory response. Recent discoveries of subsets of T cells such as T_regs and their specific functions in several regeneration competent tissue niches have opened up immense possibilities to understand human diseases where T_regs might be implicated more clearly. This model offers real‐time imaging, and therefore the use of the full animal is an excellent tool to visualize the in vivo interaction of a pathogen within the system. Also, the genomic responses of adult zebrafish tissues can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. Immune response and signaling have been well preserved all the way through evolution and mammal and zebrafish genomic responses after lipopolysaccharide (LPS) stimulation are highly correlated. In order to maintain homeostasis and regenerate various tissues, zT_regs produce growth factors that selectively stimulate the regeneration of tissue‐specific cell types. The extraordinary ability of zebrafish to regenerate numerous organs suggests that studying zT_reg activity in additional regeneration situations and clarifying the mechanism for proregenerative zT_reg development may provide insights into how human T_regs may be specifically targeted to aid regeneration of damaged tissues. Therefore, zebrafish would become a promising and potent futuristic model to review the fundamental mechanisms of T_reg biology, inflammation and regeneration and to model human inflammatory, autoimmune diseases to harness immune cell‐based therapies targeting T_reg.

CONFLICTS OF INTEREST

The authors state no conflicts of interest.

AUTHOR CONTRIBUTIONS

S.P.H. conceptualized and designed the manuscript. SG., S.A. and S.P.H. wrote the manuscript and prepared the figure.

Gupta S, Adhikary S, Hui SP. Decoding the proregenerative competence of regulatory T cells through complex tissue regeneration in zebrafish. Clin Exp Immunol. 2021;206:344–353. 10.1111/cei.13661

DATA AVAILABILITY STATEMENT

Not applicable.