Yasuhiro Mouri

Development of Autoimmunity against Transcriptionally Unrepressed Target Antigen in the Thymus of Aire-Deficient Mice

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjögren’s syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, α-fodrin. Remarkably, transcriptional expression of α-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.

Alteration of intra-pancreatic target-organ specificity by abrogation of Aire in NOD mice

Factors that determine the spectrum of target organs involved in autoimmune destruction are poorly understood. Although loss of function of autoimmune regulator (AIRE) in thymic epithelial cells is responsible for autoimmunity, the pathogenic roles of AIRE in regulating target-organ specificity remain elusive. In order to gain insight into this issue, we have established NOD mice, an animal model of type 1 diabetes caused by autoimmune attack against beta cell islets, in which Aire has been abrogated. Remarkably, acinar cells rather than beta cell islets were the major targets of autoimmune destruction in Aire-deficient NOD mice, and this alteration of intra-pancreatic target-organ specificity was associated with production of autoantibody against pancreas-specific protein disulfide isomerase (PDIp), an antigen expressed predominantly by acinar cells. Consistent with this pathological change, the animals were resistant to the development of diabetes. The results suggest that Aire not only is critical for the control of self-tolerance but is also a strong modifier of target-organ specificity through regulation of T cell repertoire diversification. We also demonstrated that transcriptional expression of PDIp was retained in the Aire-deficient NOD thymus, further supporting the concept that Aire may regulate the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus.

Biphasic Aire expression in early embryos and in medullary thymic epithelial cells before end-stage terminal differentiation

The roles of autoimmune regulator (Aire)–expressing medullary thymic epithelial cells (mTECs) in the organization of the thymic microenvironment for establishing self-tolerance are enigmatic. We sought to monitor the production and maintenance of Aire-expressing mTECs by a fate-mapping strategy in which bacterial artificial chromosome transgenic (Tg) mice expressing Cre recombinase under the control of the Aire regulatory element were crossed with a GFP reporter strain. We found that, in addition to its well recognized expression within mature mTECs, Aire was expressed in the early embryo before emergence of the three germ cell layers. This observation may help to explain the development of ectodermal dystrophy often seen in patients with AIRE deficiency. With the use of one Tg line in which Cre recombinase expression was confined to mTECs, we found that Aire+CD80high mTECs further progressed to an Aire−CD80intermediate stage, suggesting that Aire expression is not constitutive from after its induction until cell death but instead is down-regulated at the beginning of terminal differentiation. We also demonstrated that many mTECs of Aire-expressing lineage are in close contact with thymic dendritic cells. This close proximity may contribute to transfer of tissue-restricted self-antigens expressed by mTECs to professional antigen-presenting cells.

Essential Role of IκB Kinase α in Thymic Organogenesis Required for the Establishment of Self-Tolerance

IκB kinase (IKK) α exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-κB activation pathway. The in vivo function of IKKα, however, still remains elusive. Because a natural strain of mice with mutant NF-κB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKα axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKα-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKα-dependent manner was associated with defective processing of NF-κB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKα in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

Lymphotoxin Signal Promotes Thymic Organogenesis by Eliciting RANK Expression in the Embryonic Thymic Stroma

It has recently become clear that signals mediated by members of the TNFR superfamily, including lymphotoxin-β receptor (LTβR), receptor activator for NF-κB (RANK), and CD40, play essential roles in organizing the integrity of medullary thymic epithelial cells (mTECs) required for the establishment of self-tolerance. However, details of the mechanism responsible for the unique and cooperative action of individual and multiple TNFR superfamily members during mTEC differentiation still remain enigmatic. In this study, we show that the LTβR signal upregulates expression of RANK in the thymic stroma, thereby promoting accessibility to the RANK ligand necessary for mTEC differentiation. Cooperation between the LTβR and RANK signals for optimal mTEC differentiation was underscored by the exaggerated defect of thymic organogenesis observed in mice doubly deficient for these signals. In contrast, we observed little cooperation between the LTβR and CD40 signals. Thus, the LTβR signal exhibits a novel and unique function in promoting RANK activity for mTEC organization, indicating a link between thymic organogenesis mediated by multiple cytokine signals and the control of autoimmunity.

Targeted deletion of the murine corneodesmosin gene delineates its essential role in skin and hair physiology

Controlled proteolytic degradation of specialized junctional structures, corneodesmosomes, by epidermal proteases is an essential process for physiological desquamation of the skin. Corneodesmosin (CDSN) is an extracellular component of corneodesmosomes and, although considerable debate still exists, genetic studies have suggested that the CDSN gene in the major psoriasis-susceptibility locus (PSORS1) may be responsible for susceptibility to psoriasis, a human skin disorder characterized by excessive growth and aberrant differentiation of keratinocytes. CDSN is also expressed in the inner root sheath of hair follicles, and a heterozygous nonsense mutation of the CDSN gene in humans is associated with scalp-specific hair loss of poorly defined etiology. Here, we have investigated the pathogenetic roles of CDSN loss of function in the development of skin diseases by generating a mouse strain with targeted deletion of the Cdsn gene. Cdsn-deficient mouse skin showed detachment of the stratum corneum from the underlying granular layer and/or detachment within the upper granular layers due to the disrupted integrity of the corneodesmosomes. When grafted onto immunodeficient mice, Cdsn-deficient skin showed rapid hair loss together with epidermal abnormalities resembling psoriasis. These results underscore the essential roles of CDSN in hair physiology and suggest functional relevance of CDSN gene polymorphisms to psoriasis susceptibility.

Temporal Lineage Tracing of Aire-Expressing Cells Reveals a Requirement for Aire in Their Maturation Program

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7–8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80high mTECs seen in such mice. Of interest, increased CD80high mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.

Androgen Receptor Promotes Sex-Independent Angiogenesis in Response to Ischemia and Is Required for Activation of Vascular Endothelial Growth Factor Receptor Signaling

Background— Hypoandrogenemia is associated with an increased risk of ischemic diseases. Because actions of androgens are exerted through androgen receptor (AR) activation, we studied hind-limb ischemia in AR knockout mice to elucidate the role of AR in response to ischemia. Methods and Results— Both male and female AR knockout mice exhibited impaired blood flow recovery, more cellular apoptosis, and a higher incidence of autoamputation after ischemia. In ex vivo and in vivo angiogenesis studies, AR-deficient vascular endothelial cells showed reduced angiogenic capability. In ischemic limbs of AR knockout mice, reductions in the phosphorylation of the Akt protein kinase and endothelial nitric oxide synthase were observed despite a robust increase in hypoxia-inducible factor 1&agr; and vascular endothelial cell growth factor (VEGF) gene expression. In in vitro studies, siRNA-mediated ablation of AR in vascular endothelial cells blunted VEGF-stimulated phosphorylation of Akt and endothelial nitric oxide synthase. Immunoprecipitation experiments documented an association between AR and kinase insert domain protein receptor that promoted the recruitment of downstream signaling components. Conclusions— These results document a physiological role of AR in sex-independent angiogenic potency and provide evidence of novel cross-talk between the androgen/AR signaling and VEGF/kinase insert domain protein receptor signaling pathways.

NF-κB–Inducing Kinase in Thymic Stroma Establishes Central Tolerance by Orchestrating Cross-Talk with Not Only Thymocytes but Also Dendritic Cells

Essential roles of NF-κB–inducing kinase (NIK) for the development of medullary thymic epithelial cells (mTECs) and regulatory T cells have been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice). However, the exact mechanisms underlying the defect in thymic cross-talk leading to the breakdown of self-tolerance in aly mice remain elusive. In this study, we demonstrated that production of regulatory T cells and the final maturation process of positively selected conventional αβ T cells are impaired in aly mice, partly because of a lack of mature mTECs. Of note, numbers of thymic dendritic cells and their expression of costimulatory molecules were also affected in aly mice in a thymic stroma–dependent manner. The results suggest a pivotal role of NIK in the thymic stroma in establishing self-tolerance by orchestrating cross-talk between mTECs and dendritic cells as well as thymocytes. In addition, we showed that negative selection was impaired in aly mice as a result of the stromal defect, which accounts for the development of organ-specific autoimmunity through a lack of normal NIK.

Which Model Better Fits the Role of Aire in the Establishment of Self-Tolerance: The Transcription Model or the Maturation Model?

The discovery of Aire-dependent transcriptional control of many tissue-restricted self-antigen (TRA) genes in thymic epithelial cells in the medulla (medullary thymic epithelial cells, mTECs) 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. From a mechanistic viewpoint, there are two possible models to explain the function of Aire in this action. In the first model, TRAs are considered to be the direct target genes of Aire’s transcriptional activity. In this scenario, the lack of Aire protein within cells would result in the defective TRA gene expression, 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 TRA gene expression 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 TRA gene expression. In this brief review, we will focus on these two contrasting models for the roles of Aire in controlling the expression of TRAs within mTECs.