Anton M. Jetten

Essential autocrine regulation by IL-21 in the generation of inflammatory T cells

After activation, CD4+ helper T (TH) cells differentiate into distinct effector subsets that are characterized by their unique cytokine expression and immunoregulatory function. During this differentiation, TH1 and TH2 cells produce interferon-γ and interleukin (IL)-4, respectively, as autocrine factors necessary for selective lineage commitment. A distinct TH subset, termed THIL-17, TH17 or inflammatory TH (THi), has been recently identified as a distinct TH lineage mediating tissue inflammation. TH17 differentiation is initiated by transforming growth factor-β and IL-6 (refs 5–7) and reinforced by IL-23 (ref. 8), in which signal transduction and activators of transcription (STAT)3 and retinoic acid receptor-related orphan receptor (ROR)-γ mediate the lineage specification. TH17 cells produce IL-17, IL-17F and IL-22, all of which regulate inflammatory responses by tissue cells but have no importance in TH17 differentiation. Here we show that IL-21 is another cytokine highly expressed by mouse TH17 cells. IL-21 is induced by IL-6 in activated T cells, a process that is dependent on STAT3 but not ROR-γ. IL-21 potently induces TH17 differentiation and suppresses Foxp3 expression, which requires STAT3 and ROR-γ, which is encoded by Rorc. IL-21 deficiency impairs the generation of TH17 cells and results in protection against experimental autoimmune encephalomyelitis. IL-21 is therefore an autocrine cytokine that is sufficient and necessary for TH17 differentiation, and serves as a target for treating inflammatory diseases.

T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORα and RORγ

T cell functional differentiation is mediated by lineage-specific transcription factors. T helper 17 (Th17) has been recently identified as a distinct Th lineage mediating tissue inflammation. Retinoic acid receptor-related orphan receptor gamma (ROR gamma) was shown to regulate Th17 differentiation; ROR gamma deficiency, however, did not completely abolish Th17 cytokine expression. Here, we report Th17 cells highly expressed another related nuclear receptor, ROR alpha, induced by transforming growth factor-beta and interleukin-6 (IL-6), which is dependent on signal transducer and activator of transcription 3. Overexpression of ROR alpha promoted Th17 differentiation, possibly through the conserved noncoding sequence 2 in Il17-Il17f locus. ROR alpha deficiency resulted in reduced IL-17 expression in vitro and in vivo. Furthermore, ROR alpha and ROR gamma coexpression synergistically led to greater Th17 differentiation. Double deficiencies in ROR alpha and ROR gamma globally impaired Th17 generation and completely protected mice against experimental autoimmune encephalomyelitis. Therefore, Th17 differentiation is directed by two lineage-specific nuclear receptors, ROR alpha and ROR gamma.

Molecular Antagonism and Plasticity of Regulatory and Inflammatory T Cell Programs

Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.

Generation of T Follicular Helper Cells Is Mediated by Interleukin-21 but Independent of T Helper 1, 2, or 17 Cell Lineages

After activation, CD4(+) helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor beta (TGF-beta) or Th17-specific orphan nuclear receptors RORalpha and RORgamma in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-beta signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage.

CCR6 Regulates the Migration of Inflammatory and Regulatory T Cells

Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two T cell subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-β and requires two nuclear receptors, RORα and RORγ. Th17 cells also express the CCR6 ligand CCL20, which is induced synergistically by TGF-β and IL-6, which requires STAT3, RORγ and IL-21. Th17 cells, by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration.

Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism.

The last few years have witnessed a rapid increase in our knowledge of the retinoid-related orphan receptors RORα, -β, and -γ (NR1F1-3), their mechanism of action, physiological functions, and their potential role in several pathologies. The characterization of ROR-deficient mice and gene expression profiling in particular have provided great insights into the critical functions of RORs in the regulation of a variety of physiological processes. These studies revealed that RORa plays a critical role in the development of the cerebellum, that both RORα and RORβ are required for the maturation of photoreceptors in the retina, and that RORγ is essential for the development of several secondary lymphoid tissues, including lymph nodes. RORs have been further implicated in the regulation of various metabolic pathways, energy homeostasis, and thymopoiesis. Recent studies identified a critical role for RORγ in lineage specification of uncommitted CD4+T helper cells into Th17 cells. In addition, RORs regulate the expression of several components of the circadian clock and may play a role in integrating the circadian clock and the rhythmic pattern of expression of downstream (metabolic) genes. Study of ROR target genes has provided insights into the mechanisms by which RORs control these processes. Moreover, several reports have presented evidence for a potential role of RORs in several pathologies, including osteoporosis, several autoimmune diseases, asthma, cancer, and obesity, and raised the possibility that RORs may serve as potential targets for chemotherapeutic intervention. This prospect was strengthened by recent evidence showing that RORs can function as ligand-dependent transcription factors.

I[kgr]B[zgr] regulates TH17 development by cooperating with ROR nuclear receptors

Interleukin (IL)-17-producing helper T (TH17) cells are a distinct T-cell subset characterized by its pathological role in autoimmune diseases. IL-6 and transforming growth factor-β (TGF-β) induce TH17 development, in which the orphan nuclear receptors, RORγt and RORα, have an indispensable role. However, in the absence of IL-6 and TGF-β, the ectopic expression of RORγt or RORα leads to only a modest IL-17 production. Here we identify a nuclear IκB family member, IκBζ (encoded by the Nfkbiz gene), as a transcription factor required for TH17 development in mice. The ectopic expression of IκBζ in naive CD4+ T cells together with RORγt or RORα potently induces TH17 development, even in the absence of IL-6 and TGF-β. Notably, Nfkbiz-/- mice have a defect in TH17 development and a resistance to experimental autoimmune encephalomyelitis (EAE). The T-cell-intrinsic function of IκBζ was clearly demonstrated by the resistance to EAE of the Rag2-/- mice into which Nfkbiz-/- CD4+ T cells were transferred. In cooperation with RORγt and RORα, IκBζ enhances Il17a expression by binding directly to the regulatory region of the Il17a gene. This study provides evidence for the transcriptional mechanisms underlying TH17 development and points to a molecular basis for a novel therapeutic strategy against autoimmune disease.

The ROR nuclear orphan receptor subfamily: Critical regulators of multiple biological processes

The nuclear receptor superfamily, a group of structurally related, ligand-dependent transcription factors, includes a large number of orphan receptors for which no ligand has yet been identified. These proteins function as key regulators of many physiological processes that occur during embryonic development and in the adult. The retinoid-related orphan receptors (RORs) alpha, beta, and gamma comprise one nuclear orphan receptor gene subfamily. RORs exhibit a modular structure that is characteristic for nuclear receptors; the DNA-binding domain is highly conserved and the ligand-binding domain is moderately conserved among RORs. By a combination of alternative promoter usage and exon splicing, each ROR gene generates several isoforms that differ only in their amino terminus. RORs bind as monomers to specific ROR response elements (ROREs) consisting of the consensus core motif AGGTCA preceded by a 5-bp A/T-rich sequence. RORE-dependent transcriptional activation by RORs is cell type-specific and mediated through interactions with nuclear cofactors. RORs have been shown to interact with certain corepressors as well as coactivators, suggesting that RORs are not constitutively active but that their activity is under some regulatory control. RORs likely can assume at least two different conformations: a repressive state, which allows interaction with corepressor complexes, and an active state, which promotes binding of coactivator complexes. Whether the transition between these two states is regulated by ligand binding and/or by phosphorylation remains to be determined. Ca2+/calmodulin-dependent kinase IV (CaMKIV) can dramatically enhance ROR-mediated transcriptional activation. This stimulation involves CaMKIV-mediated phosphorylation not of RORs, but likely of specific nuclear cofactors that interact with RORs. RORalpha is widely expressed. In the cerebellum, its expression is limited to the Purkinje cells. RORalpha-/- mice and the natural RORalpha-deficient staggerer mice exhibit severe cerebellar ataxia due to a defect in Purkinje cell development. In addition, these mice have thin long bones, suggesting a role for RORalpha in bone metabolism, and develop severe atherosclerosis when placed on a high-fat diet. Expression of RORbeta is very restricted. RORbeta is highly expressed in different parts of the neurophotoendocrine system, the pineal gland, the retina, and suprachiasmatic nuclei, suggesting a role in the control of circadian rhythm. This is supported by observations showing alterations in circadian behavior in RORbeta-/- mice. RORgamma, which is most highly expressed in the thymus, plays an important role in thymopoiesis. Thymocytes from RORgamma-/- mice undergo accelerated apoptosis. The induction of apoptosis is, at least in part, due to a down-regulation of the expression of the antiapoptotic gene Bcl-XL. In addition to the thynic phenotype, RORgamma-/- mice lack lymph nodes, indicating that RORgamma is essential for lymph node organogenesis. Overexpression of RORgamma has been shown to inhibit T cell receptor-mediated apoptosis in T cell hybridomas and to repress the induction of Fas-ligand and interleukin 2. These studies demonstrate that RORs play critical roles in the regulation of a variety of physiological processes. Further characterization of the mechanisms of action of RORs will not only lead to the identification of ROR target genes and provide additional insight into their normal physiological functions, but will also determine their roles in disease.

Possible role of retinoic acid binding protein in retinoid stimulation of embryonal carcinoma cell differentiation

RETINOIDS, naturally occurring and synthetic analogues of vitamin A, are involved in the maintenance and differentiation of epithelial tissue1–3. Furthermore, it has been reported that these compounds inhibit the growth of certain nontransformed and virally and chemically transformed cell lines4,5. Recently, it has been shown that retinoic acid stimulates the differentiation of embryonal carcinoma cells6,7, undifferentiated early embryo-like cells present in teratocarcinomas. Embryonal carcinoma cell lines differ widely in their capacity to differentiate: a distinction has been made between ‘pluripotent’ and ‘nullipotent’ cell lines8,9. In contrast to the latter, Pluripotent embryonal carcinoma cells differentiate when injected into mice8 or when cell aggregates are formed in vitro10; some cell lines differentiate quite readily even when grown in monolayer culture. We have shown7 that retinoic acid not only stimulates the differentiation of Pluripotent embryonal carcinoma cell lines, but also induces differentiation of the so-called nullipotent embryonal carcinoma cell lines nulli SCC1 and 6050 AJ. We show here that this effect is not limited to retinoic acid but that several other retinoids have the ability to stimulate differentiation of at least one embryonal carcinoma cell line, PCC4.aza1R. Furthermore, we present evidence to suggest that the action of retinoids may be mediated by a retinoic acid binding protein.

Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ

In this paper we examined the effects of transforming growth factor beta (TGF beta) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGF beta inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGF beta causes cells to accumulate in the G0/G1 phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGF beta induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGF beta enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGF beta induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGF beta, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGF beta in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGF beta, whereas growth of one carcinoma cell line was stimulated by TGF beta. These results indicate that a modified response to TGF beta could be one mechanism involved in the aberrant growth control of malignant cells.