c-Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice.

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel-/- ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel-/- mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel-/- tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.

Extracellular Release of Antigen by Dendritic Cell Regurgitation Promotes B Cell Activation through NF-κB/cRel.

Dendritic cells (DCs) are professional APCs, which sample Ags in the periphery and migrate to the lymph node where they activate T cells. DCs can also present native Ag to B cells through interactions observed both in vitro and in vivo. However, the mechanisms of Ag transfer and B cell activation by DCs remain incompletely understood. In this study, we report that murine DCs are an important cell transporter of Ag from the periphery to the lymph node B cell zone and also potent inducers of B cell activation both in vivo and in vitro. Importantly, we highlight a novel extracellular mechanism of B cell activation by DCs. In this study, we demonstrate that Ag released upon DC regurgitation is sufficient to efficiently induce early B cell activation, which is BCR driven and mechanistically dependent on the nuclear accumulation of the transcription factor NF-κB/cRel. Thus, our study provides new mechanistic insights into Ag delivery and B cell activation modalities by DCs and a promising approach for targeting NF-κB/cRel pathway to modulate the DC-elicited B cell responses.

The NF-κB transcription factor c-Rel controls host defense against Citrobacter rodentium.

Mice lacking CD4+ T cells or B cells are highly susceptible to Citrobacter rodentium infection. In this study, we show that the activity of the transcription factor c-Rel in lymphocytes is crucial for clearance of C. rodentium. Mice deficient for c-Rel fail to generate protective antibodies and to eradicate the pathogen.

Kickstarting Foxp3 with c-Rel.

In this issue of Immunity, reports by Long et al. (2009) and Ruan et al. (2009) suggest that the transcription factor NF-kappaB-c-Rel is an important molecular mechanism by which T cell receptor-specificity for self-antigens instructs the selection of Foxp3(+) regulatory T cells.

Cutting edge: NF-κB p65 and c-Rel control epidermal development and immune homeostasis in the skin.

Psoriasis is an inflammatory skin disease in which activated immune cells and the proinflammatory cytokine TNF are well-known mediators of pathogenesis. The transcription factor NF-κB is a key regulator of TNF production and TNF-induced proinflammatory gene expression, and both the psoriatic transcriptome and genetic susceptibility further implicate NF-κB in psoriasis etiopathology. However, the role of NF-κB in psoriasis remains controversial. We analyzed the function of canonical NF-κB in the epidermis using CRE-mediated deletion of p65 and c-Rel in keratinocytes. In contrast to animals lacking p65 or c-Rel alone, mice lacking both subunits developed severe dermatitis after birth. Consistent with its partial histological similarity to human psoriasis, this condition could be prevented by anti-TNF treatment. Moreover, regulatory T cells in lesional skin played an important role in disease remission. Our results demonstrate that canonical NF-κB in keratinocytes is essential for the maintenance of skin immune homeostasis and is protective against spontaneous dermatitis.

c-Rel is Required for the Induction of pTregs in the Eye but Not in the Gut Mucosa.

Regulatory T (Treg) cells play an integral role in maintaining immune homeostasis and preventing autoimmune diseases. Forkhead box P3 expression marks the commitment of progenitor cells to the Treg lineage. Although the essential function of the nuclear factor (NF)-κB family transcription factor c-Rel in the regulation of natural Treg cells has been firmly established, little is known about whether c-Rel is involved in the in vivo generation of peripheral Treg cells (pTregs), which develop from mature CD4+ conventional T cells outside of the thymus. We sought to answer this question through the induction of pTregs in the eye and gut mucosa using ovalbumin-specific T cell receptor transgenic mice that do or do not express c-Rel. Our results showed that Tregs can be induced in the eye in a c-Rel-dependent manner when immune-mediated inflammation occurs. However, c-Rel is dispensable for the induction of pTregs in the gut mucosa after oral antigen administration. Thus, c-Rel may play distinct roles in regulating the development of pTregs in different organs. Abbreviations ACAID: Anterior Chamber-Associated Immune Deviation; ATF: activating transcription factor; CREB: cAMP responsive element-binding protein; DMEM: Dulbecco minimum essential medium; HBSS: Hanks Balanced Salt Solution; NFAT: Nuclear Factor of Activated T cells; PBS: Phosphate-buffered saline; PE: Phycoerythrin; WT: wild type.

T cell-intrinsic function of the noncanonical NF-κB pathway in the regulation of GM-CSF expression and experimental autoimmune encephalomyelitis pathogenesis.

The noncanonical NF-κB pathway induces processing of the NF-κB2 precursor protein p100, and thereby mediates activation of p52-containing NF-κB complexes. This pathway is crucial for B cell maturation and humoral immunity, but its role in regulating T cell function is less clear. Using mutant mice that express a nonprocessible p100, NF-κB2(lym1), we show that the noncanonical NF-κB pathway has a T cell-intrinsic role in regulating the pathogenesis of a T cell-mediated autoimmunity, experimental autoimmune encephalomyelitis (EAE). Although the lym1 mutation does not interfere with naive T cell activation, it renders the Th17 cells defective in the production of inflammatory effector molecules, particularly the cytokine GM-CSF. We provide evidence that p52 binds to the promoter of the GM-CSF-encoding gene (Csf2) and cooperates with c-Rel in the transactivation of this target gene. Introduction of exogenous p52 or GM-CSF to the NF-κB2(lym1) mutant T cells partially restores their ability to induce EAE. These results suggest that the noncanonical NF-κB pathway mediates induction of EAE by regulating the effector function of inflammatory T cells.

IκBε is a key regulator of B cell expansion by providing negative feedback on cRel and RelA in a stimulus-specific manner.

The transcription factor NF-κB is a regulator of inflammatory and adaptive immune responses, yet only IκBα was shown to limit NF-κB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in the regulation of B cell proliferation and survival. Loss of IκBε resulted in increased B cell proliferation and survival in response to both antigenic and innate stimulation. NF-κB activity was elevated during late-phase activation, but the dimer composition was stimulus specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers also were elevated. The corresponding dimer-specific sequences were found in the promoters of hyperactivated genes. Using a mathematical model of the NF-κB-signaling system in B cells, we demonstrated that kinetic considerations of IκB kinase-signaling input and IκBε's interactions with RelA- and cRel-specific dimers could account for this stimulus specificity. cRel is known to be the key regulator of B cell expansion. We found that the RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling revealed that the inflammatory cytokine IL-6 was hyperactivated in IκBε(-/-) B cells. When IL-6R was blocked, LPS-responsive IκBε(-/-) B cell proliferation was reduced to near wild-type levels. Our results provide novel evidence for a critical role for immune-response functions of IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel- and RelA-containing NF-κB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative-feedback regulators.

NF-κB is crucial in proximal T-cell signaling for calcium influx and NFAT activation.

In the accepted model of T-cell activation, parallel signal-transduction pathways activate the transcription factors NF-κB, NFAT, and AP-1 to drive clonal expansion of T cells in response to Ag. Genome-wide transcriptional profiling following Ag-induced CD8(+) T-cell activation in C57BL/6 mouse T cells revealed that genes regulated by NFAT were also reduced in the absence of NF-κB p50 and cRel subunits. Importantly, p50(-/-) cRel(-/-) CD8(+) T cells had significantly diminished NFAT and AP-1 activation compared with WT or PKCθ(-/-) CD8(+) T cells. Attenuated NFAT activation after TCR engagement was associated with reduced calcium influx, PLCγ and Zap70 activation. Interestingly, pharmacological bypass of PLCγ-regulated pathways largely rescued p50(-/-) cRel(-/-) T-cell proliferative defects. These results indicate a crucial and unexpected requirement for NF-κB p50 and cRel subunits in proximal TCR signaling and calcium responses. They further suggest that key defects in T cells in the absence of NF-κB pathway components may be due to impaired proximal T-cell signaling.

Ontogeny and water temperature influences the antiviral response of the Pacific oyster, Crassostrea gigas.

Disease is caused by a complex interaction between the pathogen, environment, and the physiological status of the host. Determining how host ontogeny interacts with water temperature to influence the antiviral response of the Pacific oysters, Crassostrea gigas, is a major goal in understanding why juvenile Pacific oysters are dying during summer as a result of the global emergence of a new genotype of the Ostreid herpesvirus, termed OsHV-1 µvar. We measured the effect of temperature (12 vs 22 °C) on the antiviral response of adult and juvenile C. gigas injected with poly I:C. Poly I:C up-regulated the expression of numerous immune genes, including TLR, MyD88, IκB-1, Rel, IRF, MDA5, STING, SOC, PKR, Viperin and Mpeg1. At 22 °C, these immune genes showed significant up-regulation in juvenile and adult oysters, but the majority of these genes were up-regulated 12 h post-injection for juveniles compared to 26 h for adults. At 12 °C, the response of these genes was completely inhibited in juveniles and delayed in adults. Temperature and age had no effect on hemolymph antiviral activity against herpes simplex virus (HSV-1). These results suggest that oysters rely on a cellular response to minimise viral replication, involving recognition of virus-associated molecular patterns to induce host cells into an antiviral state, as opposed to producing broad-spectrum antiviral compounds. This cellular response, measured by antiviral gene expression of circulating hemocytes, was influenced by temperature and oyster age. We speculate whether the vigorous antiviral response of juveniles at 22 °C results in an immune-mediated disorder causing mortality.

Critical role of B cell lymphoma 10 in BAFF-regulated NF-κB activation and survival of anergic B cells.

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase ß, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-x(L), a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.

The increased transcriptional response and translocation of a Rel/NF-κB homologue in scallop Chlamys farreri during the immune stimulation.

The Rel/NF-κB transcription factors can function as key regulators to modulate the expression of immune-related genes in response to immune challenge or environmental stress. In the present study, a gene coding Rel/NF-κB homologue was identified from scallop Chlamys farreri (designated CfRel). Its deduced protein comprised 359 amino acids, and contained a conserved N-terminal Rel homology domain (RHD) and an IPT domain. There was an NF-κB/Rel/dorsal domain signature sequence in the RHD domain. The mRNA transcripts of CfRel could be detected in all the tested tissues including adductor muscle, mantle, gill, gonad, haemocytes, kidney and hepatopancreas, with the highest expression level in hepatopancreas. After LPS stimulation, there were two peaks of CfRel mRNA expression level in haemocytes at 6 h (25.25-fold, P < 0.05) and 24 h (59.66-fold, P < 0.05) respectively, while the mRNA expression of CfRel was only up-regulated at 3 h after PGN stimulation (2.35-fold, P < 0.05). By Western blotting technique, CfRel protein was observed in the cytoplasm and nucleus of scallop haemocytes, and its concentration in the haemocyte nucleus increased significantly at 3 h and 12 h after LPS stimulation. The noticeable NF-κB transcription activity of CfRel protein was determined by NF-κB luciferase reporter assays (122.43%, P < 0.05), and it decreased significantly (17.61%, P < 0.05) after the coexpression of scallop IκB protein. These results collectively suggested that CfRel mRNA transcripts and protein were induced by immune stimulation, and CfRel protein could extricate itself from IκB protein and transfer into the haemocyte nucleus to modulate the immune response in scallop.

A key role for NF-κB transcription factor c-Rel in T-lymphocyte-differentiation and effector functions.

The transcription factors of the Rel/NF-κB family function as key regulators of innate and adoptive immunity. Tightly and temporally controlled activation of NF-κB-signalling pathways ensures prevention of harmful immune cell dysregulation, whereas a loss of control leads to pathological conditions such as severe inflammation, autoimmune disease, and inflammation-associated oncogenesis. Five family members have been identified in mammals: RelA (p65), c-Rel, RelB, and the precursor proteins NF-κB1 (p105) and NF-κB2 (p100), that are processed into p50 and p52, respectively. While RelA-containing dimers are present in most cell types, c-Rel complexes are predominately found in cells of hematopoietic origin. In T-cell lymphocytes, certain genes essential for immune function such as Il2 and Foxp3 are directly regulated by c-Rel. Additionally, c-Rel-dependent IL-12 and IL-23 transcription by macrophages and dendritic cells is crucial for T-cell differentiation and effector functions. Accordingly, c-Rel expression in T cells and antigen-presenting cells (APCs) controls a delicate balance between tolerance and immunity. This review gives a selective overview on recent progress in understanding of diverse roles of c-Rel in regulating adaptive immunity.

Critical role of cRel subunit of NF-κB in sepsis survival.

NF-κB is a critical regulator of gene expression during severe infections. NF-κB comprises homo- and heterodimers of proteins from the Rel family. Among them, p50 and p65 have been clearly implicated in the pathophysiology of sepsis. In contrast, the role of cRel in sepsis is still controversial and has been poorly studied in single-pathogen infections. We aimed to investigate the consequences of cRel deficiency in a cecal ligation and puncture (CLP) model of sepsis. We have approached the underlying mechanisms of host defense by analyzing bacterial clearance, systemic inflammation, and the distribution of spleen dendritic cell subsets. Moreover, by using a genome-wide technology, we have also analyzed the CLP-induced modifications in gene expression profiles both in wild-type (wt) and in rel(-/-) mice. The absence of cRel enhances mortality due to polymicrobial sepsis. Despite normal pathogen clearance, cRel deficiency leads to an altered systemic inflammatory response associated with a sustained loss of the spleen lymphoid dendritic cells. Furthermore, a whole-blood microarray study reveals that the differential outcome between wt and rel(-/-) mice during sepsis is preceded by remarkable changes in the expression of hundreds of genes involved in aspects of host-pathogen interaction, such as host survival and lipid metabolism. In conclusion, cRel is a key NF-κB member required for host antimicrobial defenses and a regulatory transcription subunit that controls the inflammatory and immune responses in severe infection.

c-Rel but not NF-kappaB1 is important for T regulatory cell development.

Regulatory T (Treg) cells are crucial for maintaining peripheral tolerance and controlling T-cell responses. The generation of Treg in the thymus requires TCR triggering and CD28 costimulation. Engagement of these receptors induces a number of signalling pathways, including the activation of NF-kappaB via PKCtheta and the Bcl-10/CARMA1/MALT complex. Previous studies have shown that PKCtheta, Bcl-10 and CARMA1 are important for Treg development. It is unclear, however, whether different members of the NF-kappaB family contribute to Treg development or homeostasis. In this study, we show that Treg numbers are reduced in the absence of c-Rel but not NF-kappaB1 (p50). Furthermore, using mixed bone marrow chimeras from WT and KO animals, we demonstrate that the requirement for PKCtheta, Bcl-10 and c-Rel is T-cell intrinsic, and cannot be rescued by the presence of WT cells. Therefore, c-Rel and NF-kappaB1 have differential roles in Treg development.

c-Rel phenocopies PKCtheta but not Bcl-10 in regulating CD8+ T-cell activation versus tolerance.

Elucidating the signaling events that promote T-cell tolerance versus activation provides important insights for manipulating immunity in vivo. Previous studies have suggested that the absence of PKCtheta results in the induction of anergy and that the balance between the induction of the transcription factors NFAT, AP1 and NF-kappaB plays a key role in determining whether T-cell anergy or activation is induced. Here, we examine whether Bcl-10 and specific family members of NF-kappaB act downstream of PKCtheta to alter CD8(+) T-cell activation and/or anergy. We showed that T cells from mice deficient in c-Rel but not NF-kappaB1 (p50) have increased susceptibility to the induction of anergy, similar to T cells from PKCtheta-deficient mice. Surprisingly T cells from Bcl-10-deficient mice showed a strikingly different phenotype to the PKCtheta-deficient T cells, with a severe block in TCR-mediated activation. Furthermore, we have also shown that survival signals downstream of NF-kappaB, are uncoupled from signals that mediate T-cell anergy. These results suggest that c-Rel plays a critical role downstream of PKCtheta in controlling CD8(+) T-cell anergy induction.

c-Rel is crucial for the induction of Foxp3(+) regulatory CD4(+) T cells but not T(H)17 cells.

The NF-kappaB/Rel family member c-Rel was described to be required for the development of T(H)1 responses. However, the role of c-Rel in the differentiation of T(H)17 and regulatory CD4(+)Foxp3(+) T cells (Treg) remains obscure. Here, we show that in the absence of c-Rel, in vitro differentiation of pro-inflammatory T(H)17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c-Rel-deficient CD4(+) T cells was severely hampered and correlated to reduced numbers of Foxp3(+) T cells in vivo. Mechanistically, in vitro conversion of naive CD4(+) T cells into iTreg was crucially dependent on c-Rel-mediated synthesis of endogenous IL-2. The addition of exogenous IL-2 was sufficient to rescue the development of c-Rel-deficient iTreg. Thus, c-Rel is essential for the development of Foxp3(+) Treg but not for T(H)17 cells via regulating the production of IL-2.

c-Rel: a pioneer in directing regulatory T-cell lineage commitment?

The transcription factor Foxp3 controls the differentiation and function of Treg, but the molecular mechanisms that regulate Foxp3 transcription remain elusive. In particular, signals and factors that open and remodel the Foxp3 locus and imprint developing Treg with a stable Foxp3 phenotype are largely unknown. Two reports in this issue of the European Journal of Immunology, together with recent reports published elsewhere, demonstrate that a member of the NF-kappaB family transcription factors, c-Rel, is required for thymic differentiation of Foxp3(+) Treg. Moreover, c-Rel is shown to regulate Foxp3 transcription directly by binding to cis-regulatory elements at the Foxp3 locus upon TCR/CD28 stimulation, including the promoter and the newly identified conserved non-coding DNA sequence harboring a "permissive" chromatin status in Treg precursors. These findings collectively suggest that c-Rel may act as a pioneer transcription factor in initiating Foxp3 transcription in Treg precursors in the thymus.

The roles of c-rel and interleukin-2 in tolerance: a molecular explanation of self-nonself discrimination.

The molecular mechanisms responsible for the exquisite discrimination between self and nonself molecules have remained enigmatic despite intense investigation. However, with the availability of adequate amounts of anergic lymphocytes produced by double transgenic mice, large numbers of immature B cells from sublethaly irradiated, hematopoietically-synchronized mice, as well as critical gene-deleted mice, it has been possible for the first time to uncover plausible molecular mechanisms that lead to tolerance versus immunity. The Rel family of transcription factors is expressed at different stages of lymphocyte maturation and differentiation. C-Rel is not activated by immature lymphocytes, which undergo either anergy or apoptosis when triggered by antigen receptors, but c-Rel is activated in mature lymphocytes. Antigen receptor triggering induces c-Rel-dependent survival and proliferative genetic programs. In T cells, a critical c-Rel-dependent gene encodes the T-cell growth factor interleukin-2 (IL-2). Thus, T cells from c-Rel gene-deleted mice produce inadequate quantities of IL-2, which renders them immunocompromised and unable to mount normal T-cell proliferative and differentiative responses. In the face of absolute IL-2 deficiency from birth, severe, multiorgan autoimmunity gradually ensues. Also, with more subtle IL-2 deficiency, organ/tissue-specific autoimmune disease becomes evident. Accordingly, both c-Rel and IL-2 appear to be key molecules for tolerance versus immunity, and doubtless will become foci for continued investigation, as well as future therapeutic targets in autoimmune diseases.