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Logo of agdisAging and DiseaseAboutEditorial BoardSubmissionAchieves
Aging Dis. 2012 June; 3(3): 248–259.
Published online 2012 May 1.
PMCID: PMC3375081

Age-Related Disruption of Steady-State Thymic Medulla Provokes Autoimmune Phenotype via Perturbing Negative Selection


The thymic medulla plays an essential role in the generation of central tolerance by eliminating self-reactive T-cell clones through thymic negative selection and developing natural regulatory T cells. Age-related FoxN1 decline induces disruption of medullary thymic epithelial cells (mTECs). However, it is unknown whether this perturbs central tolerance to increase autoimmune predisposition in the elderly. Using a loxP-floxed-FoxN1 (FoxN1flox) mouse model, which exhibits a spontaneous ubiquitous deletion of FoxN1 with age to accelerate thymic aging, we investigated whether disruption of steady-state thymic medulla results in an increase of autoimmune-prone associated with age. We demonstrated age-associated ubiquitous loss of FoxN1flox-formed two-dimensional thymic epithelial cysts were primarily located in the medulla. This resulted in disruption of thymic medullary steady state, with evidence of perturbed negative selection, including reduced expression of the autoimmune regulator (Aire) gene and disrupted accumulation of thymic dendritic cells in the medulla, which are required for negative selection. These provoke autoimmune phenotypes, including increased inflammatory cell infiltration in multiple organs in these mice. This finding in an animal model provides a mechanistic explanation of increased susceptibility to autoimmunity in aged humans, although they may not show clinic manifestations without induction.

Keywords: Aging, Thymic microstructure, Central immunological tolerance, Negative selection, FoxN1 gene, Autoimmunity

Thymic microenvironments, composed primarily of an integrated three-dimensional meshwork of thymic epithelial cells (TECs), foster T-cell development by positive (leading to self-MHC restriction) and negative (resulting in elimination of self-reactive T-cell clones in order to tolerate self-antigens in the periphery) selection to shape the T-cell receptor. The TECs can be divided into cortical and medullary TECs (cTECs and mTECs), which are located in cortical and medullary regions, with each forming distinct microenvironments. The thymic medulla provides a unique microenvironment that is essential for the generation of central immune tolerance [13] through two major mechanisms: depleting self-reactive T-cell colonies (clonal deletion) by negative selection [4] and producing CD4+ natural regulatory T cells (nTregs) by “deviated development” [5, 6]. Clonal deletion is mainly dependent on co-operation of both mTECs and thymic dendritic cells (tDCs), in which mTECs serve primarily as a reservoir of peripheral tissue-specific self-antigens, while tDCs take up and present peripheral tissue-specific self-antigens in the unique microenvironment of the thymic medulla [7, 8]. The processes of peripheral tissue-specific self-antigen presentation by mTECs and antigen transfer from mTECs to tDCs are regulated by the autoimmune regulator (Aire) gene [912].

Many studies show that defects in mTECs and disruption of thymic medullary microstructure, even a reduction in the size of the medulla, can affect the generation of central tolerance by decreasing the efficiency of Aire and/or tissue-specific self-antigen presentation [1315], leading to the generation of fewer [16], or deficient CD4+nTreg cells [17], and thereby increasing the incidence of autoimmune diseases. The functional thymic epithelium is an organized three-dimensional meshwork. However, in our previous work, we found that loss of epithelial cell-autonomous gene forkhead box N1 (FoxN1) altered the TEC meshwork from a three-dimensional-stereo to a two-dimensional-polarized cystic structure [18]. Cystic thymus is usually associated with autoimmune diseases, seen in several mouse lines including, but not limited to, ALY (severe immunodeficiency) [19], NOD (insulin-dependent diabetes) [20], and NZB (lupus-like syndrome) [21] mice.

Previous findings show that expression of FoxN1 is declined in aged thymus [22, 23]. We confirmed that reduced FoxN1 expression is causally related to age-related thymic involution [23] in an accelerated thymic aging mouse model [23] established by crossbreeding a ubiquitous promoter-driven Cre-recombinase and estrogen-receptor fusion protein (uCreERT) transgenic mouse [24], which has a low-level spontaneous leakage of the CreERT transgene due to incomplete estrogen receptor (ER) blockage in vivo [25], with a loxP-floxed FoxN1(fx) knock-in mouse [26]. Aged individuals have an increased incidence of autoimmune diseases [27, 28], and many autoimmune diseases such as rheumatoid arthritis [29] and multiple sclerosis [30] are associated with age. This, at least in part, is due to aged thymic defects in the context of genetic background and microbial environmental factors.

Recent studies indicate that there is a lifelong need to establish tolerance to self [31], because T cells are continuously generated from a young to a very old age [32]. Although aged individuals have an atrophied thymus, this atrophied thymus still processes T-cell generation, commencing with receptor-negative T-cell progenitors that need to undergo positive and negative selection in TEC-constituted microenvironment. However, the aged thymic medulla is significantly different from the young one. Therefore, it is largely unknown whether the aged thymic medulla is still able to conduct normal negative selection to generate normal central tolerance, in comparison to a young thymic medulla. If not, whether the generation of central tolerance is perturbed?

In this report, using a FoxN1 decline-induced accelerated thymic aging mouse model [23], we demonstrate that age-related FoxN1 decline-formed two-dimensional thymic cysts appeared primarily in the thymic medulla, thereby resulting in reduced expression of the Aire gene and Aire-dependent peripheral tissue-specific self-antigen genes in the thymus by a mechanism of decreased gene expression due to a loss of cells. Furthermore, we found that a disorganized medulla disrupted the ability to attract thymic dendritic cells (tDCs) accumulated in the medulla. These phenotypes are associated with perturbation of negative selection, which thereby provokes autoimmune phenotypes, including increased inflammatory cell infiltration and autoantibody deposition in multiple organs, as well as increased antinuclear antibodies in the serum. We, therefore, conclude that an increased susceptibility to autoimmunity in the elderly is potentially associated with aging-induced disruption of thymic medullary microstructure in the postnatal thymus, which results mainly from a decline in FoxN1 expression.


Mice, genotyping, and animal care

The fx/fx-uCreERT mice (C57BL/6 genetic background) were generated by crossbreeding loxP-floxed FoxN1 (FoxN1flox, fx) mice, described previously [26] (the Jackson Lab #012941), with CAG-Cre/Esr1 (ubiquitous CreERT expression, uCreERT) transgenic mice (the Jackson Lab, #004682) [24]. The fx/fx-uCreERT mice undergo spontaneous FoxN1 deletion with age [23] due to incomplete estrogen receptor (ER) blockage in vivo [25]. All young fx/fx-uCreERT mice were genotyped. Primer sequences were reported previously [26]. Mouse ages are indicated in each figure legend, dividing into young (1–2 months old); early middle-aged (6–9 months old); middle-aged (±12 months old); and aged (over 18 months old) groups. Control group was wild type (WT) and fx/fx-only mice, since loxP insertion does not affect FoxN1 gene expression [26], the fx/fx-only mice without Cre-recombinase are considered same as WT mice. In some experiments, we also used fx/+-heterozygous mice for controls. Aged WT mice were purchased from the National Institute on Aging. All animal experiments were in compliance with the protocols approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center at Fort Worth, in accordance with guidelines of the National Institutes of Health.

Real-time RT-PCR

Thymic stromal cells were enriched through three-cycle enzymatic digestions using collagenase and DNase-I as previously reported [33, 34]. Total RNA from these thymic stromal cell-enriched thymic cells was reverse transcribed with the SuperScriptIII cDNA kit (Invitrogen). Real-time PCR was performed by standard techniques in a Step-One-Plus real-time PCR system (Applied Biosystems), with TaqMan-probes for Aire, Spt1, and Chrna1 (Applied Biosystems qPCR Cat# Mm00477461_m1, Mm00839568_m1, and Mm00431627_m1, respectively) or SYBR-green reagents [Insulin I, Insulin II, and I-FABP primers previously published [35]], as well as XCL1 primer [36]. Samples were normalized to 18S RNA or GAPDH internal control. The results were analyzed by the relative quantitative (RQ) gene expression ΔΔCT method, setting the value for average controls as 1.0.

H&E staining for checking lympho-infiltration

Mouse organs (the thymus, liver, lung, pancreas, brain, and salivary and lacrimal glands) from age-matched fx/fx-uCreERT mice and control mice were fixed, cut into 5 μm-thick sections, and stained with hematoxylin and eosin (H&E).

Immunofluorescence staining

To detect serum autoantibody, 6 μm cryosections of lacrimal gland, salivary gland, adrenal gland, stomach, pancreas, ovary, prostate, intestine, testicle, and eye were prepared from young RAG−/− and WT (tissues with an enhanced block) mice. After fixation in cold acetone for 5 min, sections were incubated with 0.1% NP-40/TBS for 5 min and blocked with SuperBlock (Thermo scientific #37516) for 15 min at room temperature. The slides were blocked again with Affinipure Fab Fragment Donkey Anti-Mouse IgG (H+L) (1:10) (Jackson ImmunoResearch Laboratories, #715-007-003) for 1hr at 37°C. The sections were divided into two groups: one was incubated with sera from age-matched fx/fx-uCreERT mice, while the other one was incubated with sera from control fx/fx-only mice (1:100 dilutions) at 4°C over night, then stained with Cy3-conjugated donkey anti–mouse IgG (Jackson ImmunoResearch Laboratories, #715-166-151) for 30 min at room temperature. For immunofluorescence staining of thymic tissues, thymic cryosections (6 μm) were fixed in acetone and blocked in 10% donkey serum/TBS. Primary antibodies were rabbit anti-mouse Keratin-5 (K5, Covance), rabbit anti-mouse Claudin-3,4 (Cld3,4, Invitrogen, #34–1700 and #36–4800), rat anti-mouse K8 (Troma-1 supernatant), and rabbit anti-Aire (Santa Cruz, #M-300). Secondary reagents included Cy3-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-rat IgG (Jackson ImmunoResearch Laboratories) or Alexa-Fluor-488-conjugated anti-rabbit IgG (Invitrogen).

ELISA Assays

Anti-Nuclear Antibody concentration in mouse sera was determined using an ELISA kit (Alpha Diagnostic International, #5210), following the manufacturer’s instructions. In brief, each serum sample was diluted 1:100 and incubated in an antigen coated well. Samples were run in duplicate and the data represent the mean of the two values. The second antibody was HRP-conjugated anti-mouse IgG (H/L), and the substrate was TMB (3,3′,5,5′-tetramethylbenzidine). The absorbance was measured at 450 nm with the BioTek ELx800 ELISA reader. Meanwhile, a concentration standard curve was generated using purified immunoglobulin isotypes supplied in the kit. Concentration (μg/ml) of Anti-Nuclear Antibody in mouse serum samples was calculated against this standard curve.


For evaluation of group differences, the unpaired two-tailed Student’s t-test was used assuming equal variance. Differences were considered statistically significant at values of p < 0.05.


Age-associated deletion of FoxN1flox, mediated by spontaneously leaky uCreERT, resulted in the generation of two-dimensional thymic cysts primarily in the medulla

In previous work, we demonstrated that FoxN1flox (fx) deletion mediated by uCreERT leakage in fx/fx-uCreERT mice induced thymic aging characterized by accelerated age-related thymic involution [23]. We also reported that the K14 promoter-driven Cre-recombinase-mediated FoxN1flox deletion, i.e. FoxN1 K14 knockout resulted in increased generation of two-dimensional thymic cysts in the thymic medullary region [18]. We then wanted to determine in which region, the medulla or cortex, uCreERT-induced two-dimensional thymic cysts are primarily located in the fx/fx-uCreERT mouse thymus. Therefore, we observed the thymus of early middle-aged fx/fx-uCreERT mice without tamoxifen (TM) treatment, and detected a large number of two-dimensional thymic cysts (Fig. 1) similar to our previous finding in FoxN1 K14 knockout mice with Cld3,4+ epithelial lining (Figs. 1 C and D). These two-dimensional thymic cysts were mainly located in the medullary region (Figs. 1 B and D), in spite of FoxN1flox deletion was ubiquitous in the both medulla and cortex. The results imply that ubiquitous FoxN1flox deletion-induced thymic deterioration in the postnatal thymus is either limited primarily to or originates in the medulla. This change results in the disruption of the three-dimensional medullary steady state [18].

Figure 1.
Generation of two-dimensional thymic cysts primarily in the thymic medullary region by FoxN1flox (fx) deletion through a low level spontaneous leakage of ubiquitous CreERT. (A) Representative H&E staining of early middle-aged (9-month-old) WT ...

Expression both Aire and Aire-dependent peripheral tissue-specific antigens was decreased in early middle-aged fx/fx-uCreERT mice

The medulla plays a well-characterized role in the generation of central immune tolerance through thymocyte negative selection. One of mechanisms for central tolerance generation involves the presentation of tissue-specific self-antigens, which are expressed in a promiscuous fashion [37], and is primarily controlled by the Aire (autoimmunity regulator) gene [911]. Both Aire and tissue-specific self-antigens including Aire-dependent and Aire-independent antigens are expressed in mature mTECs [9, 37, 38]. Earlier, we found that Aire gene expression was decreased in FoxN1 K14 knockout mice [18]. Here, we confirmed that Aire+ TECs were also reduced in the early middle-aged fx/fx-uCreERT mice without TM treatment (Figs. 2A and B), and expression of the Aire gene itself and most Aire-dependent tissue-specific genes were significantly decreased (Fig. 2C). The results further confirmed that age-related uCreERT-mediated FoxN1flox deletion in the postnatal thymus disrupts Aire gene expression, as well as most tissue-specific self-antigen expression, resulting from the disrupted steady-state medulla. Decreased Aire expression is unlikely due to direct interaction of FoxN1 with Aire, but probably due to gene expression in disrupted medullary epithelial cells. Therefore, we could not use defective mTECs themselves and genes, such as Keratin 5, expressed on these defective mTECs as normal controls in Figure 2. This disruption potentially perturbs negative selection in the medulla. The results coincide with findings from others [14, 15].

Figure 2.
Expression of Aire and relevant peripheral tissue-specific self-antigens. (A) Representative immunofluorescence staining of Aire (red) versus K8 (green) on cryosections shows decreased Aire expression in early middle-aged fx/fx-uCreERT mice. (B) Summarized ...

Disruption of medullary accumulation of thymic dendritic cells in early middle-aged fx/fx-uCreERT and naturally-aged WT mice

Thymic dendritic cells (tDCs) are also important for establishing central immune tolerance because in addition to presenting ubiquitous antigens, tDCs along with mTECs present an array of peripheral tissue-specific self-antigens to direct negative selection [reviewed in [2, 3, 13]] and furthermore, tDCs process and cross-present tissue-specific self-antigens to augment deletion of self-reactive T cells [39]. A recent report shows that tDC accumulation in the medulla is facilitated by XCR1 expression in tDCs and XCL1 expression in mTECs [36]. Disruption of tDC accumulation in the medulla, which is regulated by Aire, elicits autoimmune inflammatory lesions [36]. To determine whether defects in the medulla in early middle-aged fx/fx-uCreERT mice induce dysfunctional tDC accumulation in the thymic medulla, we observed tDC distribution in the thymus of these mice, and found that the distribution of tDCs was reduced in the medullary region and pronounced in the cortical region (arrows in Fig. 3A, right panel), while in the control group (Fig. 3A, left panels) most tDCs were located in the medullary region. Same phenotype was found in the naturally-aged (21-month-old WT) thymus (33) (Fig. 3B right panel), in which FoxN1 expression is decreased (22,23). In addition, we found that XCL1 expression in sorted CD45MHC-Class-II+ epithelial cells was significantly decreased in naturally-aged mice (Fig. 3C). This may account for the inability of mTECs to attract tDCs. However, a dysfunction of tDCs themselves is unlikely because we did not find a decrease in XCR1, which is expressed in tDCs, by real-time RT-PCR (data not shown).

Figure 3.
Distribution of thymic DCs in the medulla and cortex. (A) Distribution of tDCs in the early meddle-aged fx/fx-only (left panel) and fx/fx-uCreERT (right panel) mouse thymi with immunofluorescence staining of thymic mTECs (K5, green) and tDCs (CD11c, red). ...

Early middle-aged fx/fx-uCreERT mice had a significant increase in inflammatory cell infiltration in multiple organs

As FoxN1 decline induces disruption of normal thymic medullary microstructure and homeostasis along with decreased expression of Aire and most tissue-specific self-antigens, and decreased tDC accumulation in the medullary region of the thymus, we set out to determine whether these changes, which perturb negative selection, are enough to elicit autoimmune inflammatory lesions. We observed inflammatory cell infiltration in multiple organs, including the liver, lung, pancreas, brain, and salivary glands in paraffin-embedded sections with H&E staining. We found increased inflammatory cell infiltration in multiple organs in early middle-aged (6~9 months old) fx/fx-uCreERT mice (Figs. 4A–E) in terms of both inflammatory cell infiltrated areas and infiltrated cell cluster numbers (Fig. 4B), particularly in the salivary glands (71.4% positive cases), liver (57.1% positive cases), and lung (38.8% positive cases) (Fig. 4D). These results indicate that an autoimmune-phenotype does exist in early middle-aged fx/fx-uCreERT mice.

Figure 4.
Inflammatory cell infiltration in multiple organs of middle-aged fx/fx-uCreERT mice. (A) Representative H&E staining shows inflammatory cell infiltration in the livers of early middle-aged fx/fx-uCreERT (left panel) and heterozygous littermate ...

Early middle-aged fx/fx-uCreERT mice had overt autoantibody deposition in multiple organs and a significant increase in antinuclear antibodies in the sera

We also revealed a spectrum of serum autoantibody deposition by immunofluorescence staining of frozen tissue sections. Sera were collected from early middle-aged (9-month-old) fx/fx-uCreERT and age-matched fx/fx-only control mice. We found that autoantibody deposition occurred mostly in glands and the reproductive system, such as the lacrimal and salivary, the testicles and ovaries (Fig. 5). We also found that antinuclear antibodies in the sera of early middle-aged fx/fx-uCreERT mice were significantly elevated compared to age-matched fx/fx-only control mice (Fig. 6). Higher titers of antinuclear antibodies are indicative of autoimmune predisposition usually caused by disruption of central immune tolerance.

Figure 5.
Autoantibody deposition in multiple organs of early middle-aged fx/fx-uCreERT mice. Representative immunofluorescence staining shows sera (1:100 dilutions) from early middle-aged (9-month-old) fx/fx-uCreERT and age-matched fx/fx-only control mice reacted ...
Figure 6.
Antinuclear antibodies were significantly increased in the sera of middle-aged fx/fx-uCreERT mice. A summary of antinuclear antibody levels in 8~9-month-old middle-aged fx/fx-uCreERT mice (open circles) and age-matched fx/fx-only control mice ...


We previously showed that a conditional knockout of FoxN1 in the postnatal thymus causes acute deterioration mainly in the thymic medulla [26]; progressive decline of FoxN1 expression with age is causally related to age-related thymic involution [23]; and the naturally-aged atrophied thymus has a disorganized medulla [33]. We also found that deletion of FoxN1 in K14+ TECs induces generation of a large number of two-dimensional thymic cysts in the medulla [18]. However, it is unclear whether progressive ubiquitous decline (mimicking natural aging) of FoxN1 expression with increased age also induces two-dimensional thymic cysts primarily in the medulla. If the cysts are located primarily in the medulla, do these two-dimensional epithelial cysts influence the generation of central tolerance, given the role of the thymic medulla in this process? To determine these issues we used our previously reported FoxN1flox (fx) mice carrying the uCreERT transgene, which undergoes a low level of spontaneous ubiquitous deletion of FoxN1 with increased age. We found that middle-aged fx/fx-uCreERT mice had a disruption of the steady-state thymic medulla characterized by formation of a large number of two-dimensional thymic cysts in the thymic medulla, and exhibited reduced expression of the Aire gene and peripheral tissue-specific self-antigen genes. Furthermore, this disorganized thymic medulla disrupted tDC accumulation in the medulla. Therefore, it is very plausible that negative selection was perturbed in these mice because autoimmune phenotypes were provoked, which included increased inflammatory cell infiltration and autoantibody deposition in multiple organs, as well as increased antinuclear antibodies in the sera. Thus, we concluded that increased autoimmune predisposition in the elderly is possibly associated with aging-induced disruption of steady-state thymic medulla due to the decline of FoxN1 expression.

FoxN1 has distinct roles in the prenatal and postnatal thymus. In the prenatal thymus it controls both cTEC and mTEC patterning and differentiation to generate a functional three-dimensional epithelial meshwork from a two-dimensional epithelial thymic anlage [40, 41], while in the postnatal thymus it mainly controls mTEC differentiation in the medulla or cortico-medullary junction [26] to maintain epithelial homeostasis in a three-dimensional meshwork [18] and prevent age-related thymic involution [23, 42]. It is known that the FoxN1-null mutation in germline cells, which results in inborn dysfunction of the thymus with a lack of production of T cells, does not induce autoimmunity but causes primary immunodeficiency. However, a postnatal FoxN1 decline after the thymus is fully developed primarily affects the medulla, resulting in a reduction of naïve T-cell numbers and dysfunctional T-cell output. It is well-known that the thymic medulla is specialized for the generation of central immune tolerance. Negative selection takes place in the medulla to eliminate self-reactive T-cell clones from the T-cell repertoire [4]. The efficiency of negative selection is dependent on the presentation of peripheral tissue-specific self-antigens, so-called “promiscuous gene expression” [37, 43], which is, in part, regulated by the Aire gene in mTECs [38, 44]. Although postnatal mutation in FoxN1 induces deterioration of mTECs [26] and reduces Aire+mTECs (Fig. 2), it is unlikely that FoxN1 directly controls Aire expression, as Aire mRNA was found in the thymus of FoxN1-null nude mice [45]. However, the thymic medullary steady state is crucial for the efficiency of Aire expression and negative selection.

It is well-known that complete loss of the Aire gene (Airenull) causes autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy in humans [46, 47] and autoimmune phenotypes in mice [38, 48]. Even in heterozygous Aire+/− T-cell receptor transgenic mice, lack of one copy of Aire led to diminished thymic expression of both the endogenous insulin gene and the exogenous insulin transgene, resulting in a 300% increase in islet-reactive CD4+ T cells that escape thymic deletion, and dramatically increased progression toward diabetes [11], suggesting importance of dose-dependent Aire expression. However, there is insufficient information on whether thymic architecture abnormality-induced decline in Aire expression or decrease in Aire+ TEC numbers, but not null for Aire gene, can also induce autoimmune phenotypes. The autoimmune-prone NZD mouse strain was reported to have a thymic architecture abnormality-induced decline in Aire expression in the mTEC-high (MHC-class-IIhi,UEA-1hi) subset, and have systemic autoimmunity associated with age [14]. In addition to decline of Aire and tissue-specific self-antigen expression (Fig. 2), conditional deletion of FoxN1flox resulted in the depletion of mTEC-high subsets [26] and was associated with age [23], which further indicates that postnatal FoxN1 decline-induced autoimmune predisposition arises due to deterioration of postnatal mTECs, particularly the mTEC-high subset.

mTECs and tDCs are two major antigen-presenting cell types in the medullary region responsible for conducting negative selection [7, 12]. tDCs should be sparse in the cortical region [49] and accumulated in the medulla in the normal thymus. tDC accumulation in the medulla is regulated by Aire-dependent production of XCL1 expression in the mTECs and XCR1 expression in the tDCs [36]. Transfer of antigens from mTECs to tDCs requires both cell types to be in close proximity and is the regulated by Aire [12]. Decreased XCL1 expression in naturally-aged thymus (Fig. 3C) did indeed result in disrupted tDC medullary accumulation, with large numbers of tDCs present in the cortical region and/or separation of mTECs and tDCs (Fig. 3). These directly perturb negative selection since mTECs cannot efficiently transfer antigens to tDCs, and an excess of cortical tDCs unnecessarily present self-antigens to cortical thymocyte subpopulations, which do not undergo negative selection.

In summary, postnatal FoxN1 decline-induced increase in autoimmune propensity suggests that spontaneous autoimmunity arises from the thymic medullay architectural abnormalities, thereby disrupting thymic medullay steady state. The mechanism is similar to NZB mouse model [14] and other mouse models [15]. In the postnatal FoxN1 decline model, FoxN1 is not an autoimmunity susceptibility gene, but it plays an important role in postnatal TEC homeostasis to maintain postnatal thymic medullary steady state. Its decline, associated with an increase in age, induces the formation of two-dimensional thymic cysts, even in the naturally-aged thymus. The cysts were primarily located in the medulla, which disrupts the generation of central tolerance by perturbing negative selection, including a reduction of Aire+mTECs, reduction of peripheral tissue-specific self-antigen expression, and deterioration of tDC accumulation in the thymic medullary or transfer of antigens from mTECs to tDCs, thereby provoking autoimmunity. These findings revealed an indirect role of FoxN1 in the postnatal thymus, and the animal model has a potential to be used to study increased autoimmune predisposition in the elderly.


This work was supported by NIAID/NIH grants (R01AI081995 and 3R01AI081995-03S1) to D-M. S.


Author declaration:

We have no competing financial interests.


[1] Nitta T, Murata S, Ueno T, Tanaka K, Takahama Y. Thymic microenvironments for T-cell repertoire formation. Adv Immunol. 2008;99:59–94. [PubMed]
[2] Kyewski B, Klein L. A central role for central tolerance. Annu Rev Immunol. 2006;24:571–606. [PubMed]
[3] Hogquist KA, Baldwin TA, Jameson SC. Central tolerance: learning self-control in the thymus. Nat Rev Immunol. 2005;5:772–782. [PubMed]
[4] Palmer E. Negative selection--clearing out the bad apples from the T-cell repertoire. Nat Rev Immunol. 2003;3:383–391. [PubMed]
[5] Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, Fehervari Z, Shimizu J, Takahashi T, Nomura T. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev. 2006;212:8–27. [PubMed]
[6] Wirnsberger G, Hinterberger M, Klein L. Regulatory T-cell differentiation versus clonal deletion of autoreactive thymocytes. Immunol Cell Biol. 2011;89:45–53. [PubMed]
[7] Koble C, Kyewski B. The thymic medulla: a unique microenvironment for intercellular self-antigen transfer. J Exp Med. 2009;206:1505–1513. [PMC free article] [PubMed]
[8] Klein L, Hinterberger M, von Rohrscheidt J, Aichinger M. Autonomous versus dendritic cell-dependent contributions of medullary thymic epithelial cells to central tolerance. Trends Immunol. 2011;32:188–193. [PubMed]
[9] Derbinski J, Gabler J, Brors B, Tierling S, Jonnakuty S, Hergenhahn M, Peltonen L, Walter J, Kyewski B. Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels. J Exp Med. 2005;202:33–45. [PMC free article] [PubMed]
[10] Villasenor J, Benoist C, Mathis D. AIRE and APECED: molecular insights into an autoimmune disease. Immunol Rev. 2005;204:156–164. [PubMed]
[11] Liston A, Gray DH, Lesage S, Fletcher AL, Wilson J, Webster KE, Scott HS, Boyd RL, Peltonen L, Goodnow CC. Gene dosage--limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity. J Exp Med. 2004;200:1015–1026. [PMC free article] [PubMed]
[12] Hubert FX, Kinkel SA, Davey GM, Phipson B, Mueller SN, Liston A, Proietto AI, Cannon PZ, Forehan S, Smyth GK, Wu L, Goodnow CC, Carbone FR, Scott HS, Heath WR. Aire regulates transfer of antigen from mTEC to dendritic cells for induction of thymic tolerance. Blood. 2011. [PubMed]
[13] Gallegos AM, Bevan MJ. Central tolerance: good but imperfect. Immunol Rev. 2006;209:290–296. [PubMed]
[14] Fletcher AL, Seach N, Reiseger JJ, Lowen TE, Hammett MV, Scott HS, Boyd RL. Reduced thymic Aire expression and abnormal NF-kappaB2 signaling in a model of systemic autoimmunity. J Immunol. 2009;182:2690–2699. [PubMed]
[15] Rucci F, Poliani PL, Caraffi S, Paganini T, Fontana E, Giliani S, Alt FW, Notarangelo LD. Abnormalities of thymic stroma may contribute to immune dysregulation in murine models of leaky severe combined immunodeficiency. Front Immunol. 2011;2 [PMC free article] [PubMed]
[16] Fontenot JD, Dooley JL, Farr AG, Rudensky AY. Developmental regulation of Foxp3 expression during ontogeny. J Exp Med. 2005;202:901–906. [PMC free article] [PubMed]
[17] Lomada D, Liu B, Coghlan L, Hu Y, Richie ER. Thymus medulla formation and central tolerance are restored in IKKalpha-/- mice that express an IKKalpha transgene in keratin 5+ thymic epithelial cells. J Immunol. 2007;178:829–837. [PubMed]
[18] Guo J, Rahman M, Cheng L, Zhang S, Tvinnereim A, Su DM. Morphogenesis and maintenance of the 3D thymic medulla and prevention of nude skin phenotype require FoxN1 in pre- and post-natal K14 epithelium. J Mol Med. 2011;89:263–277. [PMC free article] [PubMed]
[19] Nakamura Y, Qu N, Terayama H, Naito M, Yi SQ, Moriyama H, Itoh M. Structure of thymic cysts in congenital lymph nodes-lacking mice. Anat Histol Embryol. 2008;37:126–130. [PubMed]
[20] Nabarra B, Andrianarison I. Thymus reticulum of autoimmune mice. 3. Ultrastructural study of NOD (non-obese diabetic) mouse thymus. Int J Exp Pathol. 1991;72:275–287. [PubMed]
[21] Nabarra B, Andrianarison I. Ultrastructural studies of thymic reticulum: I. Epithelial [corrected] component. Thymus. 1987;9:95–121. [PubMed]
[22] Ortman CL, Dittmar KA, Witte PL, Le PT. Molecular characterization of the mouse involuted thymus: aberrations in expression of transcription regulators in thymocyte and epithelial compartments. Int Immunol. 2002;14:813–822. [PubMed]
[23] Sun L, Guo J, Brown R, Amagai T, Zhao Y, Su DM. Declining expression of a single epithelial cell-autonomous gene accelerates age-related thymic involution. Aging Cell. 2010;9:347–357. [PMC free article] [PubMed]
[24] Hayashi S, McMahon AP. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol. 2002;244:305–318. [PubMed]
[25] Matsuda T, Cepko CL. Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A. 2007;104:1027–1032. [PubMed]
[26] Cheng L, Guo J, Sun L, Fu J, Barnes PF, Metzger D, Chambon P, Oshima RG, Amagai T, Su DM. Postnatal tissue-specific disruption of transcription factor FoxN1 triggers acute thymic atrophy. J Biol Chem. 2010;285:5836–5847. [PMC free article] [PubMed]
[27] Prelog M. Aging of the immune system: a risk factor for autoimmunity? Autoimmun Rev. 2006;5:136–139. [PubMed]
[28] Taub DD, Longo DL. Insights into thymic aging and regeneration. Immunol Rev. 2005;205:72–93. [PubMed]
[29] Goronzy JJ, Shao L, Weyand CM. Immune aging and rheumatoid arthritis. Rheum Dis Clin North Am. 2010;36:297–310. [PMC free article] [PubMed]
[30] Zhang H, Podojil JR, Luo X, Miller SD. Intrinsic and induced regulation of the age-associated onset of spontaneous experimental autoimmune encephalomyelitis. J Immunol. 2008;181:4638–4647. [PMC free article] [PubMed]
[31] von Boehmer H, Melchers F. Checkpoints in lymphocyte development and autoimmune disease. Nat Immunol. 2010;11:14–20. [PubMed]
[32] Aw D, Silva AB, Palmer DB. Is thymocyte development functional in the aged? Aging (Albany NY) 2009;1:146–153. [PMC free article] [PubMed]
[33] Gui J, Zhu X, Dohkan J, Cheng L, Barnes PF, Su DM. The aged thymus shows normal recruitment of lymphohematopoietic progenitors but has defects in thymic epithelial cells. Int Immunol. 2007;19:1201–1211. [PubMed]
[34] Gray DH, Chidgey AP, Boyd RL. Analysis of thymic stromal cell populations using flow cytometry. J Immunol Methods. 2002;260:15–28. [PubMed]
[35] Danzl NM, Donlin LT, Alexandropoulos K. Regulation of medullary thymic epithelial cell differentiation and function by the signaling protein Sin. J Exp Med. 2010. [PMC free article] [PubMed]
[36] Lei Y, Ripen AM, Ishimaru N, Ohigashi I, Nagasawa T, Jeker LT, Bosl MR, Hollander GA, Hayashi Y, Malefyt Rde W, Nitta T, Takahama Y. Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development. J Exp Med. 2011;208:383–394. [PMC free article] [PubMed]
[37] Derbinski J, Schulte A, Kyewski B, Klein L. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat Immunol. 2001;2:1032–1039. [PubMed]
[38] Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, von Boehmer H, Bronson R, Dierich A, Benoist C, Mathis D. Projection of an immunological self shadow within the thymus by the aire protein. Science. 2002;298:1395–1401. [PubMed]
[39] Gallegos AM, Bevan MJ. Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation. J Exp Med. 2004;200:1039–1049. [PMC free article] [PubMed]
[40] Nehls M, Kyewski B, Messerle M, Waldschutz R, Schuddekopf K, Smith AJ, Boehm T. Two genetically separable steps in the differentiation of thymic epithelium. Science. 1996;272:886–889. [PubMed]
[41] Rodewald HR. Thymus organogenesis. Annu Rev Immunol. 2008;26:355–388. [PubMed]
[42] Zook EC, Krishack PA, Zhang S, Zeleznik-Le NJ, Firulli AB, Witte PL, Le PT. Overexpression of Foxn1 attenuates age-associated thymic involution and prevents the expansion of peripheral CD4 memory T cells. Blood. 2011. [PubMed]
[43] Klein L, Kyewski B. “Promiscuous” expression of tissue antigens in the thymus: a key to T-cell tolerance and autoimmunity? J Mol Med. 2000;78:483–494. [PubMed]
[44] Anderson MS, Su MA. Aire and T cell development. Curr Opin Immunol. 2010;23:198–206. [PMC free article] [PubMed]
[45] Dooley J, Erickson M, Roelink H, Farr AG. Nude thymic rudiment lacking functional foxn1 resembles respiratory epithelium. Dev Dyn. 2005;233:1605–1612. [PubMed]
[46] Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Heino M, Krohn KJ, Lalioti MD, Mullis PE, Antonarakis SE, Kawasaki K, Asakawa S, Ito F, Shimizu N. Positional cloning of the APECED gene. Nat Genet. 1997;17:393–398. [PubMed]
[47] Aaltonen J, Björses P, Perheentupa J, Horelli−Kuitunen Nina, Palotie Aarno, Peltonen Leena, Su Lee Yeon, Francis Fiona, Henning Steffen, Thiel Cora, Leharach H, Yaspo ML. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet. 1997;17:399–403. [PubMed]
[48] Liston A, Lesage S, Wilson J, Peltonen L, Goodnow CC. Aire regulates negative selection of organ-specific T cells. Nat Immunol. 2003;4:350–354. [PubMed]
[49] Kurobe H, Liu C, Ueno T, Saito F, Ohigashi I, Seach N, Arakaki R, Hayashi Y, Kitagawa T, Lipp M, Boyd RL, Takahama Y. CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance. Immunity. 2006;24:165–177. [PubMed]

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