The autoimmune regulator (Aire)-directed ectopic expression of tissue-specific antigens (TSAs) by mature medullary thymic epithelial cells (mTECs) has been viewed as an essential mechanism in the induction of central tolerance. Recent data suggest that the survival of mTECs extends beyond the Aire+ cell population to form the post-Aire mTEC population and Hassall’s corpuscles (HCs). The nature and function of these post-Aire epithelial cells and structures, however, have remained unidentified. In this study, we characterized in detail the end-stage development of mTECs and HCs in both Aire-sufficient and Aire-deficient mice. In addition, using a transgenic mouse model in which the LacZ reporter gene is under the control of the endogenous Aire promoter, we purified and analyzed the post-Aire mTECs to characterize their function. We showed that the end-stage maturation of mTECs closely resembles that of keratinocytes and that the lack of Aire results in a marked block of mTEC differentiation, which is partially overcome by ligands for RANK and CD40. We also provide evidence that, during mTEC development, Aire is expressed only once and during a limited 1–2 day period. The following loss of Aire expression is accompanied by a quick downregulation of MHC class II and CD80, and of most of the Aire-dependent and Aire-independent TSAs, with the exception of keratinocyte-specific genes. In the final stage of maturation, the mTECs lose their nuclei to become HCs and specifically express desmogleins (DGs) 1 and 3, which, via cross-presentation by APCs, may contribute to tolerance against these pemphigus vulgaris-related TSAs.

Plants frequently experience heat ramps of various severities, but how and to what degree plant metabolic activity recovers from mild and severe heat stress is poorly understood. In this study, we exposed the constitutive terpene emitter, Solanum. lycopersicum leaves to mild (37 and 41 °C), moderate (46 °C) and severe (49 °C) heat ramps of 5 min. and monitored foliage photosynthetic activity, lipoxygenase pathway volatile (LOX), and mono-and sesquiterpene emissions and expression of two terpene synthase genes, β-phellandrene synthase and (E)-β-caryophyllene/α-humulene synthase, through a 24 h recovery period upon return to pre-stress conditions. Leaf monoterpene emissions were dominated by β-phellandrene and sesquiterpene emissions by (E)-β-caryophyllene, and thus, these two terpene synthase genes were representative for the two volatile terpene classes. Photosynthetic characteristics partly recovered under moderate heat stress, and very limited recovery was observed under severe stress. All stress treatments resulted in elicitation of LOX emissions that declined during recovery. Enhanced mono-and sesquiterpene emissions were observed immediately after the heat treatment, but the emissions decreased even to below the control treatment during recovery between 2-10 h, and raised again by 24 h. The expression of β-phellandrene and (E)-β-caryophyllene synthase genes decreased between 2-10 h after heat stress, and recovered to pre-stress level in mild heat stress treatment by 24 h. Overall, this study demonstrates a highly sensitive heat response of terpenoid synthesis that is mainly controlled by gene level responses under mild stress, while severe stress leads to non-recoverable declines in foliage physiological and gene expression activities.

Autoimmune regulator (Aire) has a unique expression pattern in thymic medullary epithelial cells (mTECs), in which it plays a critical role in the activation of tissue-specific antigens. The expression of Aire in mTECs is activated by receptor activator of nuclear factor κB (RANK) signaling; however, the molecular mechanism behind this activation is unknown. Here, we characterize a conserved noncoding sequence 1 (CNS1) containing two NF-κB binding sites upstream of the Aire coding region. We show that CNS1-deficient mice lack thymic expression of Aire and share several features of Aire-knockout mice, including downregulation of Aire-dependent genes, impaired terminal differentiation of the mTEC population, and reduced production of thymic Treg cells. In addition, we show that CNS1 is indispensable for RANK-induced Aire expression and that CNS1 is activated by NF-κB pathway complexes containing RelA. Together, our results indicate that CNS1 is a critical link between RANK signaling, NF-κB activation, and thymic expression of Aire.Keywords: Autoimmune regulator · Enhancer · NF-κB · Receptor activator of nuclear factor κB · Thymic medulla See accompanying Commentary by Mitsuru MatsumotoAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCentral tolerance is achieved in the thymus by selection of a T-cell repertoire that is nonreactive to self. In the thymus, developing T cells, termed thymocytes, interact with cortical thymic epithelial cells and thymic medullary epithelial cells (cTECs and mTECs) and dendritic cells that present antigens during the positive and negative selection processes [1,2]. Due to their unique ability to express a wide range of peripheral tissue-specific antigens (TSAs), mTECs are believed to play a central role in the negative selection of self-reactive thymocytes and thereby securing tolerance to self-proteins [3,4]. The promiscuous expression of peripheral antigens in mTECs is largely controlled by autoimmune regulator Correspondence: Dr. Pärt Peterson e-mail: part.peterson@ut.ee (Aire) that constitutively promotes the transcription of hundreds of tissue-specific genes [5][6][7][8]. The importance of Aire in negative selection is exemplified by the mutations in the human Aire gene, which cause a monogenic disorder called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy [9][10][11]. Similar to human autoimmune polyendocrinopathy-candidiasisectodermal dystrophy patients, Aire-deficient mice exhibit a multiorgan autoimmune phenotype [5], although some clear differences exist including a lack of endocrine organ involvement, relatively mild disease, and the lack of high-titer neutralizing auto-Abs to type 1 interferons and interleukin 22 [12,13].The Aire protein has a unique and highly specific expression profile. Aire is rapidly upregulated for a few days [14,15] [17,24,[26][27][28]. RANK ligation activates both the classical and alternative pathways of NF-κB signaling [29]. The classical pathway involves t...

Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

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