Post-Aire Medullary Thymic Epithelial Cells and Hassall's Corpuscles as Inducers of Tonic Pro-Inflammatory Microenvironment.

While there is convincing evidence on the role of Aire-positive medullary thymic epithelial cells (mTEC) in the induction of central tolerance, the nature and function of post-Aire mTECs and Hassall's corpuscles have remained enigmatic. Here we summarize the existing data on these late stages of mTEC differentiation with special focus on their potential to contribute to central tolerance induction by triggering the unique pro-inflammatory microenvironment in the thymus. In order to complement the existing evidence that has been obtained from mouse models, we performed proteomic analysis on microdissected samples from human thymic medullary areas at different differentiation stages. The analysis confirms that at the post-Aire stages, the mTECs lose their nuclei but maintain machinery required for translation and exocytosis and also upregulate proteins specific to keratinocyte differentiation and cornification. In addition, at the late stages of differentiation, the human mTECs display a distinct pro-inflammatory signature, including upregulation of the potent endogenous TLR4 agonist S100A8/S100A9. Collectively, the study suggests a novel mechanism by which the post-Aire mTECs and Hassall's corpuscles contribute to the thymic microenvironment with potential cues on the induction of central tolerance.

Aire-expressing ILC3-like cells in the lymph node display potent APC features.

The autoimmune regulator (Aire) serves an essential function for T cell tolerance by promoting the "promiscuous" expression of tissue antigens in thymic epithelial cells. Aire is also detected in rare cells in peripheral lymphoid organs, but the identity of these cells is poorly understood. Here, we report that Aire protein-expressing cells in lymph nodes exhibit typical group 3 innate lymphoid cell (ILC3) characteristics such as lymphoid morphology, absence of "classical" hematopoietic lineage markers, and dependence on RORγt. Aire+ cells are more frequent among lineage-negative RORγt+ cells of peripheral lymph nodes as compared with mucosa-draining lymph nodes, display a unique Aire-dependent transcriptional signature, express high surface levels of MHCII and costimulatory molecules, and efficiently present an endogenously expressed model antigen to CD4+ T cells. These findings define a novel type of ILC3-like cells with potent APC features, suggesting that these cells serve a function in the control of T cell responses.

Lipopolysaccharide induces tumor necrosis factor receptor-1 independent relocation of lymphocytes from the red pulp of the mouse spleen.

It is well known that bacterial lipopolysaccharide (LPS) induces migration of several cellular populations within the spleen. However, there are no data about the impact of LPS on B and T lymphocytes present in the red pulp. Therefore, we used an experimental model in which we tested the effects of intravenously injected LPS on the molecular, cellular and structural changes of the spleen, with special reference to the red pulp lymphocytes. We discovered that LPS induced a massive relocation of B and T lymphocytes from the splenic red pulp, which was independent of the tumor necrosis factor receptor-1 signaling axis. Early after LPS treatment, quantitative real-time PCR analysis revealed the elevated levels of mRNA encoding numerous chemokines and proinflammatory cytokines (XCL1, CXCL9, CXCL10, CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, TNFα and LTα) which affect the navigation and activities of B and T lymphocytes in the lymphoid tissues. An extreme increase in mRNA levels for CCL20 was detected in the white pulp of the LPS-treated mice. The CCL20-expressing cells were localized in the PALS. Some smaller CCL20-expressing cells were evenly dispersed in the B cell zone. Thus, our study provides new knowledge of how microbial products could be involved in shaping the structure of lymphatic organs.

Irf4 Expression in Thymic Epithelium Is Critical for Thymic Regulatory T Cell Homeostasis.

The thymus is a primary lymphoid organ required for the induction and maintenance of central tolerance. The main function of the thymus is to generate an immunocompetent set of T cells not reactive to self. During negative selection in the thymus, thymocytes with autoreactive potential are either deleted or differentiated into regulatory T cells (Tregs). The molecular basis by which the thymus allows high-efficiency Treg induction remains largely unknown. In this study, we report that IFN regulatory factor 4 (Irf4) is highly expressed in murine thymic epithelium and is required to prime thymic epithelial cells (TEC) for effective Treg induction. TEC-specific Irf4 deficiency resulted in a significantly reduced thymic Treg compartment and increased susceptibility to mononuclear infiltrations in the salivary gland. We propose that Irf4 is imperative for thymic Treg homeostasis because it regulates TEC-specific expression of several chemokines and costimulatory molecules indicated in thymocyte development and Treg induction.

Pregnancy-induced thymic involution is associated with suppression of chemokines essential for T-lymphoid progenitor homing.

During normal pregnancy, the thymus undergoes a severe reduction in size and thymocyte output, which may contribute to maternal-fetal tolerance. It is presently unknown whether the pregnancy-induced thymic involution also affects nonlymphoid thymic cell populations and whether these changes in stromal cells play a role in the reduction in thymocyte numbers. Here, we characterize the changes in thymic lymphoid and nonlymphoid cells and show that pregnancy results in a reduction of all major thymic lymphoid cell populations, including the early T-lymphoid progenitors (TLPs) and thymic regulatory T cells. In addition to the thymocytes, the thymic involution also includes all major nonlymphoid cell populations, which show a profound reduction in cell numbers. We also show that during pregnancy, the thymic nonlymphoid cells exhibit decreased expression of chemokines that are essential for TLP homing: CCL25, CXCL12, CCL21, and CCL19. In addition, the expression of these chemokines was substantially downregulated by short-term treatment with progesterone but not estrogen. Collectively, these findings suggest a novel mechanism for the pregnancy-induced reduction in TLP homing and the resulting thymic involution.

TNF superfamily members play distinct roles in shaping the thymic stromal microenvironment.

The differentiation and proper function of thymic epithelial cells (TECs) depend on various tumor necrosis factor superfamily (TNFSF) signals that are needed to maintain the thymic stromal microenvironment. Nevertheless, the direct transcriptional effects of these signals on TECs remain unclear. To address this issue, we stimulated murine embryonic thymus tissue with selected TNFSF ligands and performed a gene expression profiling study. We show that Aire expression is a direct and specific effect of RANKL stimulation, whereas LTß and TNFα are major inducers of chemokines in the thymic stroma and we propose differential NF-κB binding as one possible cause of these gene expression patterns. Our work provides further insight into the complex molecular pathways that shape the thymic microenvironment and maintain central tolerance.

A highly conserved NF-κB-responsive enhancer is critical for thymic expression of Aire in mice.

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.

The many faces of aire in central tolerance.

Although the role that Autoimmune Regulator (Aire) plays in the induction of central tolerance is well known, the precise cellular and molecular mechanisms are still unclear and debated. In the prevailing view, Aire serves mainly as a direct inducer of tissue-specific antigens. However, there is a growing amount of evidence suggesting that Aire modulates the differentiation program of medullary thymic epithelial cells, which may directly contribute to the negative selection of self-reactive thymocytes. In addition, Aire has been shown to regulate the expression of many intrathymic chemokines that are required for the proper localization of thymocytes and dendritic cells, and thus are potentially important for direct and indirect self-antigen presentation in the thymic medulla. Further, recent evidence suggests that the induction of certain antigen-specific regulatory T-cells that translocate to tumors and peripheral tissues can be Aire dependent and may contribute to tissue-specific tolerance. This review summarizes the current understanding of the effects of Aire on these alternative mechanisms for the induction of Aire-induced central tolerance.

Bipotency of thymic epithelial progenitors comes in sequence.

In the thymus, in order to become MHC-restricted self-tolerant T cells, developing thymocytes need to interact with cortical and medullary thymic epithelial cells (TECs). Although the presence of a common bipotent progenitor for these functionally and structurally distinct epithelial subsets has been clearly established, the initial developmental stages of these bipotent cells have not been well characterized. In this issue of the European Journal of Immunology, Baik et al. [Eur. J. Immunol. 2013.43: 589-594] focus on the phenotypical changes of the early bipotent populations and show how the cortical and medullary markers are sequentially acquired during TEC development. These findings argue against a binary model in which both cortical and medullary lineages diverge simultaneously from lineage-negative TEC progenitors and highlight an unexpected overlap in the phenotypic properties of these bipotent TECs with their lineage-restricted counterparts.

Role of CCL19/21 and its possible signaling through CXCR3 in development of metallophilic macrophages in the mouse thymus.

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-ß receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13-CXCR5 signaling is not necessary.

Ultrastructure of medullary thymic epithelial cells of autoimmune regulator (Aire)-deficient mice.

The significance of the autoimmune regulator (Aire) transcription regulator in establishing central tolerance has recently been elucidated in great detail. Still, the role of Aire in medullary thymic epithelial cell (mTEC) physiology is not fully understood. To shed more light on this issue, we studied the ultrastructure of mTECs in Aire-deficient thymus. We show that all types of mTECs show ultrastructural signs of activation and increased intracellular traffic, which suggests that in the absence of Aire their physiology is impaired. Type 6 'large' mTECs are fully developed in Aire-deficient mice and more frequent than in the normal thymus. The frequency of type 5 'undifferentiated' mTECs is also increased. Collectively, our results suggest that the role of Aire in the physiology of mTECs could be more profound and not restricted only to the presentation of self-tissue-restricted antigens and/or apoptosis of end-stage fully mature cell types.

Autoimmune regulator deficiency results in decreased expression of CCR4 and CCR7 ligands and in delayed migration of CD4+ thymocytes.

Autoimmune regulator (Aire) has been viewed as a central player in the induction of tolerance. This study examines whether Aire can modulate the production of the thymic chemokines involved in corticomedullary migration and thus play a role in intrathymic thymocyte migration and maturation. Aire deficiency resulted in reduced gene expression and protein levels of the CCR4 and CCR7 ligands in whole thymi of mice, as determined by quantitative PCR analysis and ELISA. The expression of the CCR4 ligands coincided with Aire expression in the CD80(high) medullary thymic epithelial cells, whereas the expression of the CCR7 ligands was detected in other cell populations. Also, the expression pattern of the CCR4 and CCR7 ligands follows that of Aire during postnatal but not during embryonic development. In vitro, overexpression of Aire resulted in an up-regulation of selected CCR4 and CCR7 ligands, which induced selective migration of double-positive and single-positive CD4(+) cells. In vivo, Aire deficiency resulted in a diminished emigration of mature CD4(+) T cells from the thymi of 5-day-old mice. In conclusion, Aire regulates the production of CCR4 and CCR7 ligands in medullary thymic epithelial cells and alters the coordinated maturation and migration of thymocytes. These results suggest a novel mechanism behind the Aire-dependent induction of central tolerance.

Metallophilic macrophages are fully developed in the thymus of autoimmune regulator (Aire)-deficient mice.

Thymic metallophilic macrophages represent a significant component in the thymus physiology. Recently, we showed their presence to be dependent on functional lymphotoxin-beta receptor (LT beta R) signaling pathway. However, it is unknown whether the development of metallophilic macrophages also requires the Autoimmune regulator (Aire) transcription factor, as suggested by some studies for medullary thymic epithelial cells, or perhaps the presence of Aire-expressing thymic epithelial cells themselves. Therefore, we investigated the presence of metallophilic macrophages in Aire-deficient thymus. Our study shows that the metallophilic macrophages are fully developed in the Aire-deficient thymus; their development is not regulated via Aire transcription factor and does not require the presence of Aire-expressing epithelial cells. On the contrary, in alymphoplasia (ALY) mice (deficient in nuclear factor-kappaB-inducing kinase, NIK), which we used as negative control, thymic metallophilic macrophages are completely lacking, similarly as in LT beta R-deficient animals. Together, these results show that the development/maintenance of thymic metallophilic macrophages is executed via LT beta R circumventing the Aire transcription factor. Thus, we shed a new light on the molecular requirements for development of these cells and also show that LT beta R pathway is a common developmental regulator of metallophilic macrophages in different lymphatic organs (i.e., thymus and spleen).

Modulation of Aire regulates the expression of tissue-restricted antigens.

Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.

AIRE's CARD revealed, a new structure for central tolerance provokes transcriptional plasticity.

Developing T cells encounter peripheral self-antigens in the thymus in order to delete autoreactive clones. It is now known that the autoimmune regulator protein (AIRE), which is expressed in thymic medullary epithelial cells, plays a key role in regulating the thymic transcription of these peripheral tissue-specific antigens. Mutations in the AIRE gene are associated with a severe multiorgan autoimmune syndrome (APECED), and autoimmune reactivities are manifest in AIRE-deficient mice. Functional AIRE protein is expressed as distinct nuclear puncta, although no structural basis existed to explain their relevance to disease. In addressing the cell biologic basis for APECED, we made the unexpected discovery that an AIRE mutation hot spot lies in a caspase recruitment domain. Combined homology modeling and in vitro data now show how APECED mutations influence the activity of this transcriptional regulator. We also provide novel in vivo evidence for AIRE's association with a global transcription cofactor, which may underlie AIRE's focal, genome-wide, alteration of the transcriptome.

Cigarette smoke inhibits lipopolysaccharide-induced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells.

Chronic smoking is characterized by immunosuppressive changes in the airways, leading to chronic colonization with bacteria, which in turn may contribute to the chronic obstructive pulmonary disease. The mechanisms causing this immunosuppression, however, are poorly characterized. This study evaluated whether cigarette smoke can inhibit endotoxin (LPS)-induced inflammatory cytokine production in bronchial epithelial cells and, if so, what the mechanisms are behind this effect. Pretreatment with cigarette smoke extract (CSE) concentration dependently inhibited the LPS-induced GM-CSF and IL-8 protein release, which was accompanied by decreased expression of mRNA in human bronchial epithelial cells (Beas-2B). The increase of neutrophil chemotaxis induced by conditioned medium from LPS-treated Beas-2B cells was also suppressed by CSE. In addition, the activity of LPS-induced transcription factor AP-1, but not NF-kappaB, was down-regulated by CSE. Notably, at the concentrations used, CSE had no effect on number or viability of Beas-2B cells. These data indicate that cigarette smoke possesses immunosuppressive properties by down-regulating the bacterial pathogen-induced neutrophil-mobilizing cytokine production via suppression of AP-1 activation in the airways. Hence, this study suggests a novel mechanism by which cigarette smoke may contribute to chronic colonization and chronic obstructive pulmonary disease in smokers.

Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways.

We have previously demonstrated that administration of the recently described cytokine IL-17 in rat airways in vivo recruits and activates neutrophils locally. In the current study, we examined whether endogenous IL-17 is involved in mediating neutrophil recruitment caused by endotoxin exposure in mouse airways. Our in vivo data show that local endotoxin exposure causes the release of free, soluble IL-17 protein 6 h later. Systemic pretreatment with a neutralizing anti-IL-17 Ab almost completely inhibits neutrophil recruitment 24 h, but not 6 h, after endotoxin exposure in the airways. Pretreatment with neutralizing anti-IL-6 and anti-macrophage inflammatory protein (MIP)-2 Abs inhibits neutrophil recruitment caused by local endotoxin exposure and IL-17, respectively. Our in vitro data show that endotoxin exposure stimulates the release of soluble IL-17 protein in T lymphocytes harvested from lung and spleen, respectively, and that this cytokine release requires coculture with airway macrophages. Intracellular IL-17 protein is detected in T lymphocytes from spleen but not in airway macrophages after coculture and stimulation of these two cell types. Finally, anti-IL-17 does not alter endotoxin-induced release of IL-6 and MIP-2 from T lymphocytes and airway macrophages in coculture. In conclusion, our results indicate that endotoxin exposure causes the release of IL-17 from T lymphocytes and that this cytokine release requires the presence of macrophages. Once released, endogenous IL-17 acts in part by inducing local release of neutrophil-mobilizing cytokines such as IL-6 and MIP-2, from nonlymphocyte, nonmacrophage cells, and this contributes to recruitment of neutrophils in the airways. These IL-17-related mechanisms constitute potential targets for pharmacotherapy against exaggerated neutrophil recruitment in airway disease.

Pharmacological modulation of interleukin-17-induced GCP-2-, GRO-alpha- and interleukin-8 release in human bronchial epithelial cells.

The cytokine interleukin-17 may play a role in the recruitment of airway neutrophils, and interleukin-17 protein is increased in the airways of patients with asthma. In this study, we characterised the effect of interleukin-17 on the release of the neutrophil-recruiting cytokines granulocyte chemotactic protein (GCP)-2, growth-related oncogene (GRO)-alpha and interleukin-8 in human bronchial epithelial (HBE) cells. We also characterised the involvement of mitogen-activated protein (MAP) kinases as well as the effect of beta-adrenoceptor and glucocorticoid receptor stimulation and calcineurin and P-glycoprotein inhibition on these epithelial responses to interleukin-17. We found that interleukin-17 (1-1000 ng/ml) increased the release of GCP-2, GRO-alpha and interleukin-8 in a concentration-dependent manner. This interleukin-17-induced release of C-X-C chemokines was sensitive to inhibition of the p38 MAP kinase pathway and to stimulation of glucocorticoid receptors. In contrast, stimulation of beta-adrenoceptors increased the release of interleukin-8 and did not markedly alter the release of GCP-2 and GRO-alpha. Inhibition of calcineurin and of P-glycoproteins did not exert any substantial effect on the release of C-X-C chemokines. In conclusion, interleukin-17 bears the potential to increase neutrophil recruitment into the airways by releasing several, different C-X-C chemokines, including GCP-2, GRO-alpha and interleukin-8 in human bronchial epithelial cells. Inhibition of the p38 MAP kinase pathway and glucocorticoid receptor stimulation constitute two credible therapeutic strategies against this interleukin-17-induced release of neutrophil-recruiting cytokines.