Editorials & Opinion
Galen (129 – 210 or 216 AD) first described an organ located behind the sternum and named it ‘thymus’ because of its close resemblance with a leaf of the thyme plant. For Galen, the thymus was the ‘seat of soul, eagerness, and fortitude’, and this old misconception most probably explains why some terms like ‘troubles thymiques’ are still used in the French medical language to designate mood disorders such as those observed in unipolar and bipolar depressive diseases. Jacobus Berengarius Carpensis (1460-1530) then provided the first complete anatomical description of the thymus in his work entitled « Anatomia Carpi. Isagoge breves perlucide ac uberime, in Anatomiam humani corporis ». For a very long time, the thymus was considered as a useless vestigial organ that had become redundant during both phylogeny and human ontogeny after puberty. It is only in the early 1900s’ that J. August Hammar initiated in Sweden true biomedical researches focusing on this organ . His pioneering work was followed by numerous studies that highlighted the important neuroendocrine regulation of the thymus, in particular by the hypothalamo-hypophysial axis, thyroid hormones, adrenal and sex steroids. For a long time, the thymus was considered as a gland and therefore an intrinsic component of the endocrine system until the elucidation of its fundamental role in immunity .
Starting from 1959, one can distinguish the following milestones leading to our current knowledge in thymus physiology:
- Role of the thymus in mouse leukemia and in T-cell development [3,4].
- Developmental biology of and self-recognition by differentiating T cells in the thymus [5,6].
- Promiscuous expression by thymic epithelium of genes encoding neuroendocrine-related and peripheral tissue-restricted antigens [7-10].
- Identification of the autoimmune regulatory (Aire) gene/protein as a transcription-like factor controlling intrathymic promiscuous gene expression [11,12].
- Intrathymic selection of self-antigen specific natural regulatory T cells (nTreg) [13-15].
- Embryology of the thymus and deciphering of the lympho-stromal interactions required for intrathymic T-cell differentiation [16-18].
In all living species, the neuroendocrine and innate immune systems have coexisted and still coexist without any apparent problem (Fig. 1).
Figure 1. Integrated evolution of the neuroendocrine, innate and RAG-dependent adaptive immunity. Essential components of the neuroendocrine system have been established long ago and did not display important variation during evolution besides gene duplication or differential RNA splicing. The appearance of RAG-dependent adaptive immunity in jawed vertebrates was associated with a high risk of autotoxicity directed against the neuroendocrine system. Of note, the first thymus emerged quite concomitantly in jawed fishes, and the intrathymic presentation of neuroendocrine-related genes may be viewed a posteriori as a very efficient and economic way in instructing the adaptive T-cell system to tolerate neuroendocrine antigens as early as during intrathymic T-cell differentiation. (Adapted from Litman GW, Cannon JP, Dishaw LJ. Reconstructing immune phylogeny: new perspectives. Nat Rev Immunol 2005; 5: 866-79). Abbreviations: VCBP, variable-region-containing chitin-binding protein; VLR, variable lymphocyte receptor; TCR, T-cell receptor.
Indeed, Toll-like receptors (TLRs) that are the most important mediators of innate immunity do not have the capacity of reaction against normal self. Some anticipatory immune responses already existed in jawless vertebrates (agnathans), and were mediated by diverse variable lymphocyte receptors (VLRs) with 4-12 leucine-rich repeat modules assembled by a gene conversion process. Some 450-500 millions years ago, the emergence of transposon-like recombination activating genes RAG1and RAG2 in jawed vertebrates (gnathostomes) promoted the development of adaptive immunity [19-21]. The appearance of RAG1 and RAG2 in the genome of jawed vertebrates (most putatively via horizontal transmission), and the subsequent appearance of the combinatorial immune system, has been erroneously assimilated to the immunology’s ‘Big Bang’.
Gene recombination in somatic lymphoid cells is responsible for the random generation of diverse immune receptors for antigens, B-cell- (± 5 x 1013 BCR combinations) and T-cell receptors (± 1018 TCR combinations). Because of its inherent risk of autotoxicity (and an indemonstrable hypothetic role in the Silurian-Ordovician mass extinction of species around the same period), the emergence of this sophisticated type of immune response exerted an evolutive pressure so powerful that, in compliance with Paul Ehrlich’s concept and prediction of ‘horror autotoxicus’, novel structures and mechanisms appeared with a specific function in the setting-up of immunological self-tolerance.
Of note, the first thymus appeared in cartilaginous-jawed fishes but was preceded by thymus-like lympho-epithelial structures in the gill baskets of lamprey larvae as very recently demonstrated . These structures named ‘thymoids’ are expressing FOXN4, the orthologue of the gene encoding forkhead box N1 (FOXN1). FOXN1 is the transcription factor specific for the differentiation of thymus epithelium in jawed vertebrates, and Foxn1 mutation is responsible for the nude phenotype in mouse. Thus, FOXN1 stands at a crucial place in the development of thymus epithelium that is an absolute requirement for T-cell differentiation. Moreover, the same study has provided strong evidence for a functional analogy between VLR assembly in these thymoids and TCR recombination in the thymus. This seminal discovery opens the question about the potential existence of autoimmune-like responses in jawless vertebrates.
Two essential and closely associated mechanisms are responsible for ensuring the thymus-dependent central arm of self-tolerance: 1) negative selection of self-reactive T cells that are stochastically generated by recombinase-dependent generation of TCR diversity in the thymus (recessive tolerance), and 2) positive selection of self-specific nTreg, which are able to inactivate in periphery self-reactive T cells having escaped thymic negative selection (dominant tolerance). Today, the major unresolved question is to understand how the same associations of self-antigens and thymic major histocompatibility complex (MHC) proteins are able to mediate both dominant and recessive self-tolerance .
Another question has long concerned the biochemical nature of self that is presented in the thymus to differentiating T cells during fetal life. Since its formulation some 75 years ago, ‘self’ has been a seminal word coined in immunological language as a fecund metaphor with some equivocal correlations to philosophy and neurocognitive sciences. For unknown reasons, there was no serious attempt to elucidate the precise identity of self before a series of consecutive studies in the late 1980s and in the 1990s. Our personal contribution in this field was to define the biochemical nature of the neuroendocrine self.
First, thymic neuroendocrine self-antigens usually correspond to peptide sequences that have been mostly conserved throughout evolution of their related protein family. Second, a hierarchy characterizes their expression profile in the thymus as one dominant member synthesized in thymus epithelium represents its related neuroendocrine family in front of differentiating T lymphocytes (i.e. oxytocin for the neurohypophysial family, neurokinin A for tachykinins, neurotensin for neuromedins, corticostatin for somatostatins, and insulin-like growth factor 2 [IGF-2] for the insulin family). This hierarchical pattern is meaningful because the strength of immunological tolerance to a protein is proportional to its intrathymic concentration . Third, following Aire-regulated gene transcription, the neuroendocrine precursors are not processed according to the classic model of neurosecretion, but they undergo an antigen processing for presentation by, or in association with, thymic MHC proteins. Finally, for some of them, their intrathymic expression precedes their expression in eutopic neuroendocrine glands.
This hierarchy in the organization of the thymic repertoire of neuroendocrine self-antigens is also significant from an evolutionary point of view. Since many major physiological functions had been established before the emergence of the anticipatory adaptive immune response in jawless fishes, they had to be protected from the risk of autoimmunity inherent to this type of immune lottery. Oxytocin is a neuropeptide that is closely implicated at different steps of the reproductive process, starting from social affiliation and bonding, and is thus important for preservation of animal and human species. Through its dominant expression in thymus epithelium, oxytocin is more tolerated than its neurohypophysial homologue vasopressin, which essentially controls water homeostasis.
Interestingly, rare cases of autoimmune diabetes insipidus have been repeatedly observed whereas any autoimmunity towards hypothalamic oxytocinergic neurons has never been described. A similar reasoning may be applied to the members of the insulin family, IGF-2, IGF-1 and insulin itself. There is no report of autoimmunity against IGF-2, the dominant thymic self-peptide of the insulin family during fetal life, whereas insulin is the primary autoantigen of type 1 diabetes. Because of their close homology, thymic neuroendocrine self-antigens however promote immunological cross-tolerance to their whole family and tolerance to insulin is weaker in Igf2-/- mice than in wild-type mice .
As already theorized by Burnet, the pathogenesis of autoimmune diseases may depend on a failure of self-tolerance and the development of ‘forbidden’ self-reactive immune clones . The progressive increase in immune complexity during evolution may explain why failures of self-tolerance are increasingly detected with most of them occurring in the human species. There is mounting evidence that a dysfunction in the mechanisms responsible for thymus-dependent dominant and recessive self-tolerance is playing a major role in the development of the autoimmune response toward many organs. Loss-of-function Aire single mutations are responsible for a very rare autosomal recessive disease named autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy (APECED) or autoimmune plolyglandular syndrome type 1 (APS-1).
Depending on their genetic background, Aire-/- mice exhibit several signs of peripheral autoimmunity, which are associated with a significant decrease in thymic transcription of neuroendocrine genes, including those encoding oxytocin, insulin and IGF-2 [27,28]. Our current in-depth knowledge in thymus physiology and physiopathology should translate very soon into the design of innovative tolerogenic and regulatory strategies aimed at restoring immunological tolerance that is absent or broken in autoimmunity, the heaviest price paid by the human species for preserving self so efficiently against non-self.
Immunoneuroendocrinology was recognized as a scientific field early in the 20th century, soon after Paul Ehrlich identified immunology as a specific domain of scientific investigation. By the 1930s, Hans Selye introduced the concept of stress-induced and adrenal cortex-mediated thymus involution and secondary immunosuppression. The dissection of the intricate cellular and molecular interactions between the major systems of cell-to-cell signaling — the neural, endocrine, and immune systems — was relaunched in the1980s and this scientific domain has received only gradual acceptance by the scientific community.
Endocrinologists did not hesitate to widely open the door to this new field and provided the first robust experimental arguments for its fundamental relevance to physiology. Immunoneuroendocrinology has been expanded exponentially, and the immunological self-tolerance of neuroendocrine proteins is now recognized as an obvious necessity for preserving general homeostasis of living organisms. Indeed, all hormones and neuropeptides exert an important control upon the immune and inflammatory responses through binding to and activation of neuroendocrine receptors expressed by immunocompetent cells. If self-tolerance to neuroendocrine ligands and receptors were not firmly installed, then the risk of developing autoimmune phenomena would be extremely high and species survival would be severely compromised.
Aging of the immune system (immunosenescence) is characterized by a higher susceptibility to infections, an increase in the incidence of cancer, as well as a decrease in response to vaccines. Although thymopoiesis (generation of naïve T cells) is maintained until late in life, thymus adipose involution has been long considered as ‘the’ hallmark of immunosenescence. Thymic involution is associated with a marked decrease in the generation of diverse T cells (in particular naïve CD4+ T cells), an expansion of memory CD8+ T cells, and a diminished influence of thymus-dependent central self-tolerance. Involution of the thymus after hypophysectomy was one of the first evidences for the control of the immune system by a neuroendocrine structure .
Numerous studies have unambiguously demonstrated that the antehypophysial growth hormone (GH) is able to reverse the age-dependent involution of the thymus [30-32]. Today, the restoration of thymus function appears as an important objective in the elderly, as well as in patients suffering with AIDS or several hematological diseases . It can now be anticipated that GH, IGF-1, GH secretagogues (such as ghrelin), GH and ghrelin receptor agonists, as well as other thymus-specific growth factors will be used in the near future for regenerating thymopoiesis and thymus tolerogenic function as well as, secondarily, several immune functions including responses to vaccines in aged and other immunocompromised patients.
The studies of Dr. Geenen’s research team are or have been supported by FRS, the Fund for Research in Industry and Agronomy (FRIA, Belgium), by the Fund Leon Fredericq for biomedical research at the University Hospital of Liege, by the Special Research Fund of the University of Liege, by the Walloon Region (Tolediab, Senegene and Raparray projects), by the Belgian Association of Diabetes, by the European Commission (Eurothymaide FP6 Integrated Project, www.eurothymaide.org), by the Juvenile Diabetes Research Foundation (JDRF, New York, USA) and by the European Association for the Study of Diabetes (EASD, Düsseldorf, Germany).
Vincent Geenen is research director at the Fund of Scientific Research (FRS, Belgium), professor of Developmental biology and History of biomedical research at the University of Liege (Belgium), and Clinical Head at the division of Endocrinology at the University Hospital of Liege.
My gratitude is due to Max D. Cooper, my mentor at the American Association of Immunologists, for recent stimulating e-mail discussions.
University of Liege Center of Immunology, Institute of Pathology CHU-B23, B-4000 Liege-Sart Tilman, Belgium
V Geenen – Research Director at Belgian F.S.R.-NFSR, University of Liege, GIGA-I3 — Center of Immunology (CIL), CHU-B34, B-4000 Liege-Sart Tilman, Belgium
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