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Keywords:

  • Promiscuous gene expression;
  • Self-tolerance;
  • Thymic organogenesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

The current prevailing view regarding the role of Aire in self-tolerance is that it is involved in the transcriptional control of many tissue-restricted self-antigen genes in thymic epithelial cells in the medulla (mTECs); however, accumulating evidence also suggests that Aire has other roles, e.g. in mTEC differentiation, and furthermore that Aire can either promote or inhibit the mTEC differentiation program, i.e. Aire does not play a neutral role in mTEC differentiation. This review discusses when and how Aire plays an important role in controlling the organization of mTECs required for the expression of self-antigen genes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

The mechanisms underlying the autoimmune pathology caused by Aire deficiency are a focus of intense research aimed at answering the fundamental question of how the immune system discriminates between self and non-self within the thymic microenvironment 1. The discovery of Aire-dependent transcriptional control of many tissue-restricted self-antigen (TRA) genes in thymic epithelial cells (TECs) in the medulla (mTECs), where Aire is most strongly expressed 2, has raised the intriguing question of how the single Aire gene can influence the transcription of such a large number of TRA genes within mTECs 3–5. Transcriptional control of TRA gene expression by Aire has drawn considerable attention since the original landmark report describing Aire regulation 2. In addition, the recent emergence of a novel epigenetic feature of Aire's action on TRA gene expression – plant homeodomain (PHD) 1 of Aire preferentially binds with unmethylated form of histone3 lysin 4 (H3K4me0) – has stimulated further efforts in this field 6, 7. However, the functional significance of Aire in the differentiation program of mTECs is equally noteworthy when considering how mTECs gain, in an Aire-dependent manner, the ability to express a large battery of TRA genes that are authentically expressed by parenchymal organs, a phenomenon termed promiscuous gene expression (PGE). In this brief review, I will address these topics by highlighting two contrasting models proposed for when and how Aire controls the differentiation program of mTECs.

Link between the mTEC differentiation program and TRA gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

Cortical TECs (cTECs) and mTECs have previously been indicated to derive from a common bipotent progenitor 8, 9, whereas one recent report suggests that the thymus contains a small population of epithelial stem cells that conserve the capacity to integrate in a complex three-dimensional network organized in cTECs and mTECs 10. Although it is now widely accepted that mTECs are clonally derived 11, there has been active debate as to how the mTECs acquire their unique ability for PGE; one model, the terminal differentiation model, suggests that PGE by mTECs is a specialized property attained upon terminal differentiation. In contrast, another model, the developmental model, suggests that PGE by mTECs reflects a general developmental process that occurs in any authentic tissues with high levels of cellular turnover, broad developmental potential, and complex environmental cues that can direct individual mTEC precursors into various developmental pathways 1, 12. Although the distinction between these two models is of interest, the focus of this brief review is Aire and its influence on PGE and/or thymic organogenesis rather than mTEC differentiation per se (see Table 1A).

Table 1. Summary of the models describing aspects regarding PGE, Aire and thymic organogenesis
AspectComment
A: How do mTECs acquire their unique ability for PGE?
Terminal differentiation modelAlthough this aspect is related to concepts discussed, the topic itself is beyond the scope of this brief review. The details of these models are discussed in other reviews such as 12.
Developmental model 
B: How does Aire control the PGE and/or thymic organogenesis?
Transcription model: Aire is a component of the transcriptional machinery for PGEAlthough opinion seems to favor the transcription model over the maturation model, this brief review focuses on the significance of the maturation model.
Maturation model: Aire is an agent for differentiation of Aire-expressing cell-lineage(s) 
C: What is the role of Aire in the maturation model?
Interruption of the mTEC differentiation program (Model 1: only an absence of Aire would reveal the full program of mTEC terminal differentiation)This is the main topic of this brief review, highlighting these two contrasting models.
Promotion of the mTEC differentiation program (Model 2: defective accomplishment of the mTEC differentiation program in the absence of Aire) 

From a mechanistic viewpoint, there are two possible models to explain the function of Aire in the PGE and/or thymic organogenesis required for establishment of self-tolerance (Table 1B). In the first model, Aire may play a tolerogenic role by transcriptionally activating TRA genes within the type(s) of mTECs characterized by Aire expression. In this scenario, the lack of Aire protein within cells would impair their tolerogenic function due to the defective PGE, while the maturation program of mTECs would be unaffected in principle. The second model hypothesizes that Aire is necessary for the maturation program of mTECs. In this case, we assume that the mTEC compartment does not mature normally in the absence of Aire. If acquisition of the properties of PGE depends on the maturation status of mTECs, a defect of such an Aire-dependent maturation program in Aire-deficient mTECs can also result in impaired PGE. For clarity, I term these two models the transcription model (i.e. direct transcriptional control of TRA genes by Aire) and the maturation model (i.e. control of maturation of Aire-expressing cell-lineage(s) by Aire). To put this concept in a more extreme way, the transcription model considers that Aire is a component of the transcriptional machinery for PGE, whereas in the maturation model, Aire is an agent of mTEC differentiation. Currently, opinion seems to favor the transcription model over the maturation model because of the significant changes in TRA gene expression seen in mTECs in Aire-deficient mice.

Beyond TRA gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

In comparison with the remarkable changes noted in the expression profiles of TRA genes in Aire-deficient mTECs 2, 13, morphological alterations in the medullary components, suggestive of the maturation model, were not initially appreciated. One reason for this could be that mice deficient in several signal transducing molecules and NF-κB components downstream of TNF receptor superfamily members exhibiting dramatic organizational abnormalities, such as a small medulla, a paucity of mTECs including Aire-expressing cells, and a loss of architectural integrity of mTECs as assessed using immunohistochemistry and/or flow cytometry, were described at about the same time as the initial description of Aire-deficient mice (as reviewed in 14). Since these TNFR signal-transduction-deficient mice, in many cases, also showed a more profound defect of TRA gene expression than that seen in Aire-deficient mice, it seemed rather obvious that the defect in TRA gene expression in the TNFR signal-transduction-deficient mice was caused by the abnormal mTEC development, a situation that did not seem to apply to Aire-deficient mice. Upon subsequent detailed inspection of Aire-deficient mice, however, several indications of morphological changes in thymic organization in these mice were also revealed 15, suggesting that Aire might be involved in the differentiation program of mTECs. The first of these findings was obtained using immunohistochemistry with Ulex europaeus agglutinin 1 (UEA-1) and monoclonal antibodies, which both react with a subset of mTEC. The Aire-deficient thymus lacked clusters of mTECs strongly labeled with UEA-1, although low levels of UEA-1 staining remained 15. Staining with anti-keratin 14 (K14) mAb also demonstrated a less dense medullary compartment accompanied by a prominence of epithelial cysts in the Aire-deficient thymus 15. Importantly, these changes were observed before any autoimmune pathology had become noticeable, making it unlikely that the alterations in the medullary compartment were secondary to the onset of autoimmune disease.

Aire expression in distal stages of mTEC differentiation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

Given that Aire+CD80high and Aire+MHC IIhigh mTECs develop from AireCD80low 16, 17 and AireMHC IIlow 18 immature mTECs, respectively, and that Aire+ mTECs are postmitotic 18, it is now clear that Aire is expressed in the distal stages of mTEC differentiation. Indeed, Aire+ mTECs are negative for p63 expression, a regulator of the proximal stages of epithelial cell differentiation 19–21; however, it had not been determined in which distal stage (beginning or end) Aire was expressed. In other words, it was not clear whether Aire+CD80high mTECs undergo further differentiation accompanied by phenotypic change(s) before their cell death, or whether they maintain this cellular signature until they die. Therefore, it was necessary to determine more precisely the timing of Aire expression during the course of the mTEC differentiation program. In this respect, a fate-mapping strategy allowing permanent marking of cells expressing a gene of interest, even after extinction of its transcription, was expected to be informative (basis for fate mapping and its application to the study of thymus epithelium is reviewed in 22). In this strategy, bacterial artificial chromosome transgenic mice expressing Cre recombinase under the control of the Aire regulatory element (i.e. Aire/Cre knock-in mice) were crossed with a GFP reporter strain, thereby making it possible to mark the cells that had expressed Aire at some time during their life history with GFP 23. This situation contrasts markedly with Aire/GFP knock-in mice, in which Aire expression can be monitored on a real-time basis 19 (as described in “Two contrasting models” below). Unfortunately, specific marking of Aire-expressing cells in the thymus (Aire+ mTECs) by a fate-mapping strategy proved to be impossible because of Aire expression from epiblast of E6.5 embryos, in which thymic organogenesis has not yet begun 23; however, with the use of a particular transgenic line in which cell marking with GFP subsequent to Aire expression was confined to the mTECs, possibly due to the low Aire/Cre transgenic expression, the existence of additional differentiation stage(s) after Aire+CD80high was revealed. Namely, the Aire+CD80high stage(s) were found to be followed by extinction of Aire expression together with down-regulation of CD80 expression, thereby generating AireCD80intermediate mTECs 23. Supporting this finding, a recent study has suggested that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events controlled by distinct mechanisms; initial formation of Aire+ mTECs depends on RANK signaling provided by lymphoid-tissue inducer cells 16, whereas continued mTEC development to the stage of expression of involucrin, a marker of terminally differentiated epithelium 24 (see “Model 2: Promotion of the mTEC differentiation program by Aire”), maps to activation of the lymphotoxin pathway by mature thymocytes 25. Given that mTECs at the Aire+CD80high stage(s) are the most competent for PGE, the functional significance of mTECs at the AireCD80intermediate stage(s) needs to be determined. It is possible that Aire-expressing mTEC lineage(s) at this stage(s) contributes to central tolerance by mechanisms beyond PGE, as exemplified by antigen processing/presentation 26, cross-presentation 18, and intrathymic thymocyte migration and maturation 27.

The existence of post-Aire stage(s) during the course of mTEC differentiation is apparently inconsistent with the notion that Aire expression in mTECs results in apoptosis. Aire's proapoptotic activity has been deduced from the increased percentage of the CD80high mTEC population (in which Aire+ mTECs reside) in Aire-deficient mice, despite the lack of Aire's ability to directly cause proliferative arrest of mTECs 18. Furthermore, overexpression of Aire in an mTEC line by gene transfection causes overt apoptosis of the cells, although this effect was delayed by a couple of days after transfection 18. One possible explanation for the discrepancy may be the delayed effect which suggests that the apoptoic events initiated by Aire expression are neither rapid nor direct. Whether or not Aire exerts any proapoptotic activity in mTECs is a critical point when considering the mTEC differentiation program (as discussed in Model 1: Interruption of the mTEC differentiation program by Aire), further study is needed to establish definitively the relationship between Aire and apoptotic events in mTECs.

Two contrasting models

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

The aim of this brief review was first to focus on the significance of the role of Aire in controlling the organization of mTECs (Table 1B); however, one question arising from this is what is the outcome of the loss of Aire on thymic organogenesis (and for PGE). In other words, how does Aire regulate the mTEC differentiation program? Here I discuss the two contrasting models for this critical issue (Table 1C).

Model 1: Interruption of the mTEC differentiation program by Aire

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

TEC differentiation can be defined by the patterns of keratin expression 28; however, a widely held binary model where a K5+K8+ progenitor gives rise to K5/K14K8+ cTECs or K5/K14+K8 mTECs seems to require some revision because although K5 and K8 are expressed throughout the medulla and cortex respectively, cells coexpressing K8 with K5 and K14 are widespread throughout the medulla and not restricted to the cortico-medullary junction 15. In the medulla, two distinct types of cells have been determined 20. Stellate K5+ mTECs expressing variable levels of K8 are the predominant cell type(s), whereas a small subset of K5K8+ cells show a globular profile without any visible cellular projections 20. Interestingly, Farr and colleagues have reported that the frequency of the latter cell type is increased in Aire-deficient mice 15. The differentiation stage(s) of these globular K5K8+ mTECs has emerged as a critical question for understanding the role of Aire in mTEC differentiation. This issue has been approached from two perspectives: the proposed proapoptotic activity of Aire (as described above in “Aire expression in distal stage of mTEC differentiation”) and the epithelial differentiation program modeled by the prostate epithelium.

The program of prostatic epithelial differentiation might serve as a useful comparison for mTEC differentiation because the prostatic epithelium bears a numbers of similarities to TECs 20; basal prostatic epithelial cells express p63, high levels of K5/K14, and low levels of K8, as is the case for immature mTECs. These basal prostatic epithelial cells terminally differentiate to become luminal epithelium lacking p63 and K5/K14, while expressing high levels of K8. If this keratin expression pattern during the course of prostatic epithelial differentiation were applied to mTEC differentiation, globular mTECs with K5K8+ expression would represent end-stage terminally differentiated cells. If the model of prostatic epithelial differentiation is further combined with the notion, discussed in “Aire expression in distal stage of mTEC differentiation,” that Aire induces apoptosis in mTECs 18, then globular K5K8+ mTECs that are increased in numbers in Aire-deficient mice could be cells that have survived the stage in which Aire normally terminates the mTEC differentiation program at the K5+ (variably expressing K8) stage, exhibiting a stellate morphology 20. In this model, stellate K5+ mTECs are normally eliminated by Aire's proapoptotic activity before completion of their terminal differentiation, and only an absence of Aire would reveal the full program of mTEC terminal differentiation ending with the globular K5K8+ phenotype (Fig. 1A).

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Figure 1. Two contrasting models for the Aire-mediated mTEC differentiation program. It is known that Aire-expressing cell progenitors differentiate into AireCD80low immature mTECs, and then become Aire+CD80high mature mTECs. (A) In model 1 (Interruption of the mTEC differentiation program by Aire), stellate K5+ mTECs expressing variable levels of K8 are the predominant cell type(s), and Aire expression at this stage(s) results in apoptosis of the mTECs, thereby terminating further differentiation (upper pathway). Absence of Aire would prevent apoptosis revealing the full program of mTEC terminal differentiation ending with a globular K5K8+ phenotype (lower pathway). (B) Regarding model 2 (Promotion of the mTEC differentiation program by Aire), fate-mapping has revealed that Aire+CD80high mature mTECs differentiate further into the AireCD80intermediate stage(s) (upper pathway). Lack of Aire in mTECs results in defective accomplishment of the differentiation program, with the cells remaining at the pre-mature stage just before terminal differentiation (lower pathway). These “Aire-less” CD80high mTECs have a globular cell shape and lack the transcriptional activity for Aire-dependent TRA genes.

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Model 2: Promotion of the mTEC differentiation program by Aire

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

Lineage marking of Aire-expressing cells using a knock-in mouse strategy has led to an opposing view regarding the roles of Aire in the mTEC differentiation program 19 to that described in “Model 1: Interruption of the mTEC differentiation program by Aire.” The coding sequence of GFP was inserted into the Aire gene locus in a manner allowing concomitant disruption of functional Aire protein expression. This strategy enabled mTECs committed to expressing Aire to be distinguished from Aire-non-expressing mTECs, in both the presence (Aire+/gfp) and absence (Airegfp/gfp) of functional Aire protein. Many GFP+ “Aire-less” mTECs in Airegfp/gfp mice were detected by both immunohistochemistry and flow cytometry, indicating that the Aire protein itself is not necessary for the production of particular mTEC lineage(s) committed to expressing Aire. Remarkably, detailed analysis using immunohistochemistry demonstrated that the morphology of the GFP+ cells from the Airegfp/gfp thymus was altered in comparison with GFP+ cells containing functional Aire protein from the Aire+/gfp mouse thymus; in the Airegfp/gfp thymus, more GFP+ cells exhibited a globular shape rather than a dendritic to fibroblastic morphology, as compared with GFP+ cells from the Aire+/gfp thymus.

As for model 1, the critical question is what is the differentiation stage(s) that these cells represent. In marked contrast to Farr's model (model 1), we have suggested that GFP+ “Aire-less” globular-shaped mTECs from the Airegfp/gfp thymus correspond to mTECs that have ceased to differentiate further due to the lack of Aire, thereby remaining at the pre-mature stage just before terminal differentiation (Fig. 1B) 19; however, whether or not these cells are the same population as the globular K5K8+ mTECs pointed out by Farr and colleagues remains to be determined. Our interpretation was based in part on the general assumption that globular cell shapes reflect a less mature state than DC shapes in a given cell lineage. Further evidence that a lack of Aire in mTECs results in defective accomplishment of mTEC differentiation has emerged from studies focusing on the cell differentiation markers expressed by mTECs.

In the skin, involucrin expression is restricted to postmitotic epithelial cells and serves as a marker of epidermal and follicular terminal differentiation 24. Interestingly, immunohistochemistry of the human thymus using anti-involucrin antibody stains characteristic swirled epithelial structures known as Hassall's corpuscles 29, which is consistent with the fact that Hassall's corpuscles are composed of terminally differentiated mTECs 30. Defective accomplishment of the mTEC differentiation program in the absence of Aire is suggested by the decreased numbers of mTECs expressing involucrin, together with an absence of typical Hassall's corpuscle-like structures, in the thymus of Aire-deficient mice 19. Then, how we can integrate the findings of pre-mature cease of mTEC differentiation program in Aire-deficient mice with their defective PGE? Given that mTECs acquire the capacity for PGE by becoming differentiated, it is possible that defective PGE in Aire-deficient mice is due to failure of mTECs to mature to the stage(s) where PGE is a cell-autonomous propensity. In this regard, model 2 might be better suited for the explanation of defective PGE from Aire-deficient mTECs than that by model 1.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

I have described two contrasting models in which Aire plays an important role in controlling the mTEC differentiation program (Table 1C) based on the sole assumption that Aire expression reflects a maturation stage of mTEC differentiation, and does not reflect activation status and/or condition, although it should be noted that Aire expression is conditional at least in ES cells 23. To conclude which model is more appropriate, it will be important to determine whether Aire induces apoptosis concomitant with its expression per se, thereby inhibiting further differentiation of mTECs. It will also be important to obtain more mTEC differentiation markers by which we can determine the stage(s) of Aire-expressing cell-lineage(s) 31 that are quantitatively and/or qualitatively altered in Aire-deficient thymus. Finally, identification of Aire's target genes relevant to the Aire-mediated mTEC differentiation program is critical for obtaining a definitive answer to this question.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References

The author thanks Dr. Andrew G. Farr for critical reading of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest: The author declares no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Link between the mTEC differentiation program and TRA gene expression
  5. Beyond TRA gene expression
  6. Aire expression in distal stages of mTEC differentiation
  7. Two contrasting models
  8. Model 1: Interruption of the mTEC differentiation program by Aire
  9. Model 2: Promotion of the mTEC differentiation program by Aire
  10. Concluding remarks
  11. Acknowledgements
  12. References