The thymic microenvironment and its role in T-cell differentiation

The thymus is a primary lymphoid organ, in which bone marrow-derived T-cell precursors undergo differentiation, ultimately leading to the migration of positively selected thymocytes to the T-cell-dependent areas of peripheral lymphoid organs (see Fig. 1). Such a process involves a sequential expression of various proteins and rearrangements of the T-cell receptor (TCR) genes. Most immature thymocytes express neither the TCR complex nor the CD4 or CD8 accessory molecules; being called double-negative thymocytes, and representing 5% total thymocytes. As maturation progresses thymocytes acquire the membrane expression of the CD4 and CD8 markers, generating the CD4⁺CD8⁺ double-positive cells, which comprise 80% of the whole population. In this stage, TCR genes are rearranged, and productive rearrangements yield the membrane expression of the TCR (complexed with the CD3) in low densities (TCR^low). Thymocytes that do not undergo a productive TCR gene rearrangement die by apoptosis, whereas those expressing productive TCR interact with peptides presented by molecules of the MHC, expressed on microenvironmental cells. This interaction determines the positive and negative selection events, crucial for normal thymocyte differentiation. Negative selection results in apoptosis-mediated cell death. Positively selected thymocytes progress to the mature TCR^highCD4⁺CD8⁻ or TCR^highCD4⁻CD8⁺ single positive stage, comprising 15% thymocytes that ultimately leave the organ to form the large majority of the peripheral T-cell repertoire (reviewed in⁽Reference Ciofani and Zúñiga-Pflücker⁴⁾).

Thymocyte differentiation occurs as cells migrate within the thymic lobules: TCR⁻CD4⁻CD8⁻ and TCR⁺CD4⁺CD8⁺ cells are cortically located, whereas mature TCR⁺CD4⁺CD8⁻ and TCR⁺CD4⁻CD8⁺ thymocytes are found in the medulla. Along with this journey, thymocytes interact with various components of the thymic microenvironment, a three-dimensional network formed of thymic epithelial cells (TEC), macrophages, dendritic cells, fibroblasts and extracellular matrix (ECM) components. In addition to the key interaction, involving the TCR/peptide-MHC, in the context of CD8 or CD4 molecules, the thymic microenvironment influences thymocyte maturation via adhesion molecules and ECM; interactions that are relevant to thymocyte migration⁽Reference Savino, Mendes da Cruz and Silva^5, Reference Savino, Mendes-da-Cruz and Smaniotto⁶⁾. Moreover, microenvironmental cells modulate thymocyte differentiation by soluble polypeptides, comprising: (a) cytokines, such as IL-1, IL-3, IL-6, IL-7, IL-8 and stem cell factor; (b) chemokines and (c) thymic hormones, including thymulin, thymopoietin and thymosin-α1⁽Reference Savino, Mendes da Cruz and Silva^5–Reference Petrie and Züniga-Pflucker⁸⁾.

Thymocyte development is altered in protein–energy malnutrition

As stated earlier, one of the most conspicuous changes in malnutrition is thymic atrophy, which is essentially due to a massive thymocyte death, particularly affecting the immature stage of CD4⁺CD8⁺ cells⁽Reference Chandra¹⁾. Yet, it should be pointed out that, in addition to the increase in thymocyte death seen in thymuses from malnourished individuals, there is an intrathymic decrease in cell proliferation, as ascertained by the very low numbers of cells labelled with proliferating cell nuclear antigen, a marker for cell proliferation⁽Reference Mitsumori, Takegawa and Shimo⁹⁾. Thus, protein–energy malnutrition-related thymocyte depletion results from enhanced thymocyte death plus decreased thymocyte proliferation. It should be pointed out that the major change in the thymic lymphoid compartment is also observed in human subjects suffering from malnutrition: a severe thymic atrophy with cortical thymocyte depletion is a consistent finding in necropsies of malnourished subjects⁽Reference Chandra^1, Reference Lyra, Madi and Maeda¹⁰⁾. Indeed, by means of echography, atrophy of the organ was also observed in vivo in malnourished children, as compared to age-matched healthy individuals⁽Reference Parent, Chevalier and Zalles¹¹⁾. Importantly, a study conducted in Guinea-Bissau showed that the reduction in thymus size at birth correlated with infant mortality⁽Reference Aaby, Marx and Trautner¹²⁾. Fortunately, malnutrition-induced thymic atrophy seems to be reversible if appropriate diet is provided, as it has been demonstrated in a longitudinal study carried out with Bolivian severely malnourished children who were treated for nutrition rehabilitation, and that has been accompanied by ultrasound scanning. In these malnourished children, there was a severe reduction in the thymus volume, paralleled by abnormally high proportions of circulating immature T lymphocytes and lower proportions of mature T cells. Two months after beginning the diet rehabilitation, the thymic area of these children was recovered⁽Reference Chevalier, Sevilla and Zalles¹³⁾.

Thymocyte depletion associated with deficiencies in vitamins and trace elements: the zinc-deficiency paradigm

In addition to protein-related malnutrition, trace element as well as vitamin deficiencies result in thymic atrophy, with cortical thymocyte depletion⁽Reference Kuvibidila, Dardenne and Savino^14–Reference Nodera, Yanagisawa and Wada¹⁷⁾. In Fe-deficient mice, a decrease in mitogen-induced proliferative response of thymocytes has been demonstrated⁽Reference Kuvibidila, Dardenne and Savino¹⁴⁾.

Many of the effects of protein–energy malnutrition on the immune function may be a result of associated alterations in metabolism of metals or vitamins, which in turn affect directly the immune system⁽Reference Cunningham-Rundles, McNeeley and Moon¹⁸⁾. The abnormal incidence of various infections and the existence of lymphopenia and lymphoid organ atrophy in malnourished children have been repeatedly demonstrated and evidence points to Zn insufficiency in this type of immunodeficiency⁽Reference Shankar and Prasad¹⁹⁾.

Zn plays a major role in cell division, differentiation, apoptosis and gene transcription, and strongly influences the immune system, affecting primarily T cells⁽Reference Chesters, Odel and Sunde²⁰⁾. The studies of the effects of severe Zn deficiency in experimental animals and human subjects showed substantial thymic atrophy as well as accelerated lymphopenia, leading to the reduction in cell- and antibody-mediated responses, thus influencing the susceptibility to infectious diseases⁽Reference Fraker, King, Garvy and Klurfeld^21–Reference Fraker, King and Laakko²³⁾.

Early observations showed that mice maintained on a Zn-deficient diet develop a progressive thymic involution: after 4 weeks the thymus retains only 25% of its original size and at 6 weeks only a few thymocytes remain in the organ⁽Reference Fernandes, Nair and Once²⁴⁾. Such changes are observed mostly in the thymic cortex, with a severe loss of CD4⁺CD8⁺ thymocytes, and can be reversed by Zn supplementation⁽Reference Fraker, Depascale-Jardieu and Zwickl^25, Reference King, Osati-Ashtiani and Fraker²⁶⁾. Moreover, marginal Zn deficiency, in the early post-natal period, also results in substantial reduction in thymic size⁽Reference Beach, Gershwin and Hurley²⁷⁾.

The mechanism(s) of heightened apoptosis in Zn deficiency mice remain(s) to be precisely determined. However, glucocorticoid hormones seem to be involved, since Zn deficiency yields a chronic stimulation of corticosterone production⁽Reference Fraker, Osati-Ashtiani and Wagner²⁸⁾, and adrenalectomy prevents thymic atrophy secondary to Zn deficiency.

These studies raise concern about the impact of intrathymic cell death in human subjects who are deficient in Zn due to suboptimal diet or chronic disease⁽Reference Fraker²⁹⁾. In this respect, nutritional supplementation should be considered in chronically ill patients, with compromised immune defence, as reported in AIDS patients⁽Reference Baum, Shor-Posner and Campa^30, Reference Baum, Campa and Lai³¹⁾. Zn supplementation resulted in a significant increase in CD4⁺ T cells and a decreased mortality. This notion can also be applied in Chagasic patients, since they exhibit a decrease in serum Zn concentrations⁽Reference Burguera, Burguera and Alarcon³²⁾; the same being observed in a variety of haemopoietic organs of infected rats⁽Reference Matousek de Abel de la Cruz, Burguera and Burguera³³⁾. Accordingly, the severity of experimental Chagas disease is much higher in Zn-deficient mice⁽Reference Fraker, Caruso and Kierszenbaum³⁴⁾.

Acute infections induce thymic atrophy

Severe thymic atrophy is also a common feature in acute infections, reflecting the massive depletion of CD4⁺CD8⁺cortical thymocytes (Table 1). This has been shown in a variety of infections, such as AIDS, rabies, malaria, Chagas disease and schistosomiasis, among others (reviewed in⁽³⁾). In some cases, thymocyte loss is so severe that the cortical region of thymic lobules virtually disappears, as a consequence of the CD4⁺CD8⁺ thymocyte death. Also, similar to what is seen in malnutrition, proliferative response of thymocytes from acutely infected individuals is reduced: we found a significant decrease in both concanavalin A- and anti-CD3-driven proliferative responses in murine Chagas disease. Interestingly, this was paralleled by a decrease in the intrathymic production of IL-2, a major T-cell proliferation cytokine⁽Reference Leite-de-Moraes, Minoprio and Dy³⁵⁾.

Table 1. Thymic atrophy in human subjects and experimental infectious diseases (modified from⁽Reference Savino³⁾)

SIV, Simian immunodeficiency virus; ND, not determined.

Thymocyte depletion seen in malnutrition and acute infections is partially under hormonal control

It is now well established that the physiology of the thymus (including both lymphoid and microenvironmental compartments) is influenced by a variety of hormones and neuropeptides⁽Reference Savino and Dardenne⁷⁾. It has been shown that glucocorticoid-circulating levels are increased in protein-malnourished mice, as compared to age-matched controls. Additionally, implanted corticosterone-containing pellets, able to generate glucocorticoid serum levels equivalent to those found in malnourished mice, were sufficient to yield thymocyte depletion⁽Reference Barone, O'Brien and Stevenson³⁶⁾. As discussed later, leptin also seems to be involved. It has been shown that human subjects and rodents lacking proper leptin production or expressing defective leptin receptors, bear a certain degree of immunodeficiency characterized by reduced T-cell proliferative response to various mitogens, impaired production of IL-4 and inappropriate antibody production after immunization⁽Reference Munzberg and Myers^37–Reference Farooqi, Matarese and Lord³⁹⁾. Interestingly, leptin/leptin receptor-deficient animals exhibit an atrophy of lymphoid tissues, particularly the thymus, and such a defect can be reversed by the reposition of the hormone⁽Reference Howard, Lord and Matarese⁴⁰⁾. Leptin was also able to prevent starvation-induced thymic atrophy⁽Reference Howard, Lord and Matarese^40, Reference Mito, Yoshino and Hosoda⁴¹⁾, strongly suggesting that this hormone is one mediator of malnutrition-induced thymic atrophy. It is thus conceivable that in malnutritional states, the imbalance in the production of leptin (which is decreased) and glucocorticoid hormones (which are increased) is at least partially responsible for thymocyte depletion and consequent atrophy of the organ, as we previously proposed⁽Reference Savino^42, Reference Savino, Dardenne and Veloso⁴³⁾.

The precise mechanisms responsible for the thymic atrophy seen in acute infections are not completely elucidated, and may vary in distinct diseases. But similar to malnutrition, one major pathway is related to the rise in glucocorticoid hormone levels in the blood, a classical effect comprised within the stress response of the organism to the infection. In fact, glucocorticoid serum levels are enhanced in Trypanosoma cruzi-infected mice⁽Reference Leite de Moraes, Hontebeyrie-Joskowicz and Leboulanger^44, Reference Corrêa-de-Santana, Paez-Pereda and Theodoropoulou⁴⁵⁾, and, as discussed later, are likely involved, at least partially, in the T. cruzi-induced thymic atrophy⁽Reference Perez, Bottasso and Savino⁴⁶⁾. Thymocyte depletion seen in rabies virus-infected mice⁽Reference Cardenas-Palomo, de Souza-Matos and Chaves-Leal⁴⁷⁾ can be prevented by adrenalectomy prior to infection. In murine Chagas disease, adrenalectomy alone did not prevent T. cruzi-induced cortical thymocyte depletion⁽Reference Leite de Moraes, Hontebeyrie-Joskowicz and Leboulanger⁴⁴⁾. Nevertheless, more recently it was demonstrated that a complete functional inhibition of glucocorticoid receptors by in vivo injection of RU-486, did prevent thymocyte depletion following acute T. cruzi infection⁽Reference Roggero, Pérez and Tamae-Kakazu⁴⁸⁾. Whether leptin levels are down-regulated in acutely infected levels remains to be determined and represents an interesting open field of investigation.

The thymic microenvironment is altered in malnutrition and acute infections

In addition to the lymphoid compartment, the thymic microenvironment is affected in various malnutritional and infectious conditions. Morphological changes in the thymic epithelium from protein-malnourished mice include the decrease in the volume of the epithelial tissue in the cortex and in the medulla of thymuses from malnourished mice, as compared to well-nourished control animals⁽Reference Mittal, Woodward and Chandra⁴⁹⁾. By contrast, an increase of intracytoplasmic accumulations of large, circular, homogeneously electron-dense profiles, rich in free and esterified cholesterol was reported in both cortical and medullary TEC of malnourished animals⁽Reference Mittal and Woodward⁵⁰⁾. Unfortunately, no data were reported concerning TEC death in this experimental model.

In T. cruzi acutely infected mice, we demonstrated changes in the expression of medullary and cortical-specific markers, as compared to controls, together with a general shrinkage of the thymic epithelial network⁽Reference Savino, Leite de Moraes and Hontebeyrie-Joskowicz⁵¹⁾.

Conceptually, these findings tell us that the thymic epithelium is morphologically altered in malnutrition and infection. As seen later, functional changes of the thymic epithelium are also seen in both these pathological conditions.

Changes in the patterns of thymocyte migratory responses in acute infections

In addition to the thymocyte depletion seen in several infectious diseases, changes in the migratory responses have also been observed. As mentioned earlier, thymocyte depletion parallels T. cruzi-induced alterations of the thymic microenvironment, comprising phenotypic changes and functional changes in the TEC network, with an enhancement in the deposition of cell migration-related molecules such the ECM proteins, fibronectin and laminin, as well as the chemokines CXCL12 and CCL21⁽Reference Cotta de Almeida, Mendes da Cruz and Bonomo^60, Reference Mendes-da-Cruz, Silva and Cotta-de-Almeida⁶¹⁾. These changes promote increased migratory responses to the corresponding ligands, and are likely related to the abnormal release of double-positive cells from the thymus into the periphery, resulting in more than 15-fold increase in double-positive cell numbers in subcutaneous lymph nodes. In this vein, it is noteworthy that double-positive cells seen in peripheral lymphoid organs express high densities of ECM and chemokine receptors⁽Reference Cotta de Almeida, Mendes da Cruz and Bonomo^60, Reference Mendes-da-Cruz, Silva and Cotta-de-Almeida⁶¹⁾. Among these abnormally released double-positive cells in the periphery, we found lymphocytes expressing potentially autoreactive TCR, which are normally deleted in the thymus of uninfected mice. This suggests that during the infection, immature T lymphocytes escape from the thymic central tolerance process and migrate to the lymph nodes where they eventually differentiate into mature CD4⁺ or CD8⁺ cells⁽Reference Savino^3, Reference de Meis, Morrot and Farias-de-Oliveira⁶³⁾.

In a second murine model of parasitic diseases, the thymus was evaluated in mice acutely infected with Plasmodium berghei. Again there is a thymic atrophy, with loss of cortical-medullary limits and the intrathymic presence of parasites⁽Reference Andrade, Gameiro and Nagib⁶⁴⁾. We also analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors. An increased expression of ECM components was observed in the thymus from infected mice, in parallel to a down-regulation of fibronectin and laminin receptor surface expression in thymocytes from these animals. Moreover, in the thymus from infected mice, we found increased contents of CXCL12 and CXCR4 and decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymus from infected mice were analysed. The evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative and CD4⁺ and CD8⁺ single-positive cells from P. berghei-infected mice have higher migratory responses, as compared to controls. Interestingly, increased numbers of these T-cell subpopulations were found in the spleen of infected mice, suggesting an abnormal export of T lymphocytes from the thymus of mice undergoing acute malaria infection⁽Reference Gameiro, Nagib and Andrade⁶⁵⁾.