REL and BHLHE40 Variants Are Associated with IL-12 and IL-10 Responses and Tuberculosis Risk.

The major human genes regulating Mycobacterium tuberculosis-induced immune responses and tuberculosis (TB) susceptibility are poorly understood. Although IL-12 and IL-10 are critical for TB pathogenesis, the genetic factors that regulate their expression in humans are unknown. CNBP, REL, and BHLHE40 are master regulators of IL-12 and IL-10 signaling. We hypothesized that common variants in CNBP, REL, and BHLHE40 were associated with IL-12 and IL-10 production from dendritic cells, and that these variants also influence adaptive immune responses to bacillus Calmette-Guérin (BCG) vaccination and TB susceptibility. We characterized the association between common variants in CNBP, REL, and BHLHE40, innate immune responses in dendritic cells and monocyte-derived macrophages, BCG-specific T cell responses, and susceptibility to pediatric and adult TB in human populations. BHLHE40 single-nucleotide polymorphism (SNP) rs4496464 was associated with increased BHLHE40 expression in monocyte-derived macrophages and increased IL-10 from peripheral blood dendritic cells and monocyte-derived macrophages after LPS and TB whole-cell lysate stimulation. SNP BHLHE40 rs11130215, in linkage disequilibrium with rs4496464, was associated with increased BCG-specific IL-2+CD4+ T cell responses and decreased risk for pediatric TB in South Africa. SNPs REL rs842634 and rs842618 were associated with increased IL-12 production from dendritic cells, and SNP REL rs842618 was associated with increased risk for TB meningitis. In summary, we found that genetic variations in REL and BHLHE40 are associated with IL-12 and IL-10 cytokine responses and TB clinical outcomes. Common human genetic regulation of well-defined intermediate cellular traits provides insights into mechanisms of TB pathogenesis.

Whole-genome landscape of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform-specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3'-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell-like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

The NF-κB Transcription Factor c-Rel Modulates Group 2 Innate Lymphoid Cell Effector Functions and Drives Allergic Airway Inflammation.

Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and orchestration of early type 2 immune responses. Upon tissue damage, ILC2s are activated by alarmins such as IL-33 and rapidly secrete large amounts of type 2 signature cytokines. ILC2 activation is governed by a network of transcriptional regulators including nuclear factor (NF)-κB family transcription factors. While it is known that activating IL-33 receptor signaling results in downstream NF-κB activation, the underlying molecular mechanisms remain elusive. Here, we found that the NF-κB subunit c-Rel is required to mount effective innate pulmonary type 2 immune responses. IL-33-mediated activation of ILC2s in vitro as well as in vivo was found to induce c-Rel mRNA and protein expression. In addition, we demonstrate that IL-33-mediated activation of ILC2s leads to nuclear translocation of c-Rel in pulmonary ILC2s. Although c-Rel was found to be a critical mediator of innate pulmonary type 2 immune responses, ILC2-intrinsic deficiency of c-Rel did not have an impact on the developmental capacity of ILC2s nor affected homeostatic numbers of lung-resident ILC2s at steady state. Moreover, we demonstrate that ILC2-intrinsic deficiency of c-Rel alters the capacity of ILC2s to upregulate the expression of ICOSL and OX40L, key stimulatory receptors, and the expression of type 2 signature cytokines IL-5, IL-9, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Collectively, our data using Rel-/- mice suggest that c-Rel promotes acute ILC2-driven allergic airway inflammation and suggest that c-Rel may contribute to the pathophysiology of ILC2-mediated allergic airway disease. It thereby represents a promising target for the treatment of allergic asthma, and evaluating the effect of established c-Rel inhibitors in this context would be of great clinical interest.

The c-Rel transcription factor limits early interferon and neuroinflammatory responses to prevent herpes simplex encephalitis onset in mice.

Herpes simplex virus type 1 (HSV-1) is the predominant cause of herpes simplex encephalitis (HSE), a condition characterized by acute inflammation and viral replication in the brain. Host genetics contribute to HSE onset, including monogenic defects in type I interferon signaling in cases of childhood HSE. Mouse models suggest a further contribution of immune cell-mediated inflammation to HSE pathogenesis. We have previously described a truncating mutation in the c-Rel transcription factor (RelC307X) that drives lethal HSE in 60% of HSV-1-infected RelC307X mice. In this study, we combined dual host-virus RNA sequencing with flow cytometry to explore cell populations and mechanisms involved in RelC307X-driven HSE. At day 5 postinfection, prior to HSE clinical symptom onset, elevated HSV-1 transcription was detected together with augmented host interferon-stimulated and inflammatory gene expression in the brainstems of high-responding RelC307X mice, predictive of HSE development. This early induction of host gene expression preceded pathological infiltration of myeloid and T cells in RelC307X mice at HSE onset by day 7. Thus, we establish c-Rel as an early regulator of viral and host responses during mouse HSE. These data further highlight the importance of achieving a balanced immune response and avoiding excess interferon-driven inflammation to promote HSE resistance.

Inherited human c-Rel deficiency disrupts myeloid and lymphoid immunity to multiple infectious agents.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

c-Rel Is Required for IL-33-Dependent Activation of ILC2s.

Group 2 innate lymphoid cells (ILC2s) are emerging as important cellular regulators of homeostatic and disease-associated immune processes. The cytokine interleukin-33 (IL-33) promotes ILC2-dependent inflammation and immunity, with IL-33 having been shown to activate NF-κB in a wide variety of cell types. However, it is currently unclear which NF-κB members play an important role in IL-33-dependent ILC2 biology. Here, we identify the NF-κB family member c-Rel as a critical component of the IL-33-dependent activation of ILC2s. Although c-Rel is dispensable for ILC2 development, it is critical for ILC2 function in the lung, with c-Rel-deficient (c-Rel-/- ) mice present a significantly reduced response to papain- and IL-33-induced lung inflammation. We also show that the absence of c-Rel reduces the IL-33-dependent expansion of ILC2 precursors and lower levels of IL-5 and IL-13 cytokine production by mature ILC2s in the lung. Together, these results identify the IL-33-c-Rel axis as a central control point of ILC2 activation and function.

NF-κB c-Rel Is Dispensable for the Development but Is Required for the Cytotoxic Function of NK Cells.

Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-κB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their expression. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c- Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.

METTL3 restrains papillary thyroid cancer progression via m6A/c-Rel/IL-8-mediated neutrophil infiltration.

Growing evidence indicates that N6-methyladenosine (m6A) is the most pervasive RNA modification in eukaryotic cells. However, the specific role of METTL3 in papillary thyroid carcinoma (PTC) initiation and development remains elusive. Here we found that downregulation of METTL3 was correlated with malignant progression and poor prognosis in PTC. A variety of gain- and loss-of-function studies clarified the effect of METTL3 on regulation of growth and metastasis of PTC cells in vitro and in vivo. By combining RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (meRIP-seq), our mechanistic studies pinpointed c-Rel and RelA as downstream m6A targets of METTL3. Disruption of METTL3 elicited secretion of interleukin-8 (IL-8), and elevated concentrations of IL-8 promoted recruitment of tumor-associated neutrophils (TANs) in chemotaxis assays and mouse models. Administration of the IL-8 antagonist SB225002 substantially retarded tumor growth and abolished TAN accumulation in immunodeficient mice. Our findings revealed that METTL3 played a pivotal tumor-suppressor role in PTC carcinogenesis through c-Rel and RelA inactivation of the nuclear factor κB (NF-κB) pathway by cooperating with YTHDF2 and altered TAN infiltration to regulate tumor growth, which extends our understanding of the relationship between m6A modification and plasticity of the tumor microenvironment.

Negative regulation of FOXP3 expression by c-Rel O-GlcNAcylation.

O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.

c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.

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.

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

Inhibition of Glycolysis in Pathogenic TH17 Cells through Targeting a miR -21-Peli1-c-Rel Pathway Prevents Autoimmunity.

It is well known that some pathogenic cells have enhanced glycolysis; the regulatory network leading to increased glycolysis are not well characterized. In this study, we show that CNS-infiltrated pathogenic TH17 cells from diseased mice specifically upregulate glycolytic pathway genes compared with homeostatic intestinal TH17 cells. Bioenergetic assay and metabolomics analyses indicate that in vitro-derived pathogenic TH17 cells are highly glycolytic compared with nonpathogenic TH17 cells. Chromatin landscape analyses demonstrate TH17 cells in vivo that show distinct chromatin states, and pathogenic TH17 cells show enhanced chromatin accessibility at glycolytic genes with NF-κB binding sites. Mechanistic studies reveal that miR-21 targets the E3 ubiquitin ligase Peli1-c-Rel pathway to promote glucose metabolism of pathogenic TH17 cells. Therapeutic targeting c-Rel-mediated glycolysis in pathogenic TH17 cells represses autoimmune diseases. These findings extend our understanding of the regulation TH17 cell glycolysis in vivo and provide insights for future therapeutic intervention to TH17 cell-mediated autoimmune diseases.

c-Rel gain in B cells drives germinal center reactions and autoantibody production.

Single-nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center B (GCB) cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels among B cell subsets. In c-Rel-overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program, and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.

PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation.

The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.

Cells are sensitive to changes in their environment. For example, maintaining normal salt levels in the blood, also called tonicity, is essential for the health of individual cells and the organism as a whole. Tonicity controls the movement of water in and out of the cell: high levels of salt inside the cell draw water in, while high levels of salt outside the cell draw water out. If salt levels in the environment surrounding the cells become too high, too much water will be drawn out, causing the cells to shrink. Changes in tonicity can cause the cell to become stressed. Initially, cells adapt to this stress by switching on sets of genes that help restore fluid balance and allow the cell to regain its normal shape and size. If the increase in tonicity exceeds tolerable stress levels and harms the cell, this initiates an inflammatory response which ultimately leads to cell death. However, it remained unclear how cells switch from adapting to responding with inflammation. Now, Farabaugh et al. have used an experimental system which mimics high salt to identify the mechanism that allows cells to switch between these two responses. The experiments showed that when salt levels are too high, cells switch on a stress sensing protein called PACT, which activates another protein called PKR. When PACT was deleted from mouse cells, this led to a decrease in the activity of inflammatory genes, and prevented the cells from self-destructing. Other proteins that are involved in the adaptive and inflammatory response are the NF-κB family of proteins and TonEBP. Farabaugh et al. found that under low intensity stress, when salt levels outside the cell are slightly too high, a family member of NF-κB works with TonEBP to switch on adaptive genes. But, if salt levels continue to rise, PACT activates and turns on PKR. This blocks the interaction between NF-κB and TonEBP, allowing another family member of NF-κB to interact with TonEBP instead. This switches the adaptive response off and the inflammatory response on. There are many diseases that involve changes in tonicity, including diabetes, cancer, inflammatory bowel disease, and dry eye syndrome. Understanding the proteins involved in the adaptive and inflammatory response could lead to the development of drugs that help to protect cells from stress-induced damage.

Differential Expression of Long Noncoding RNAs and Their Function-Related mRNAs in the Peripheral Blood of Allergic Rhinitis Patients.

BACKGROUND: The mechanism of long noncoding RNAs (lncRNAs) involved in the development of allergic rhinitis (AR) remains unclear. OBJECTIVE: We investigated the mechanism by which differentially expressed lncRNAs contribute to pathogenesis of AR. METHODS: Expression profiles of lncRNAs and mRNAs were analyzed by microarray detection from the blood samples of 3 AR patients and 3 control subjects, and the main lncRNAs were verified by quantitative real-time polymerase chain reaction (qRT-PCR) in the peripheral blood of 16 AR patients and 18 control subjects. GO (Gene_Ontology), Pathway, and Disease analysis of differentially expressed lncRNAs and mRNAs, and transcription factor prediction analysis were performed to explore synergistic effect of differentially expressed lncRNAs and their function-related mRNAs on AR pathogenesis. RESULTS: Thirty-one lncRNAs were differentially expressed in the peripheral blood from AR patients, and 4 of the 5 most differentially expressed lncRNAs had significantly higher levels in AR patients than in control subjects by qRT-PCR analysis. A lncRNA-mRNA coexpression network analysis identified 16 pairs of positive correlations between the 4 lncRNAs and coexpressed mRNAs. GO, Pathway, and Disease analyses indicated that the 4 lncRNAs were correlated with 7 mRNAs enriched in terms of inflammation, immune response, and allergic diseases. Transcription factor prediction results suggested that Oct-1, AP-1, NF-kappaB, and c-Rel play key roles in the pathogenesis of AR mediated by lncRNAs. CONCLUSION: Our results provide new insights into how lncRNAs and their function-related mRNAs might contribute to AR.

Integrative analysis suggests cell type-specific decoding of NF-κB dynamics.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

Constitutive expression of c-REL in uveal melanoma patients: correlation with clinicopathological parameters and patient outcome.

PURPOSE: Uveal melanoma (UM) is the most common intraocular cancer with a high mortality rate that requires new research in the field of prevention and treatment. c-REL is a member of the nuclear factor κB (NF-κB) transcription factor family and an emerging regulator of tumorigenesis. Therefore, the objective of the study is to evaluate the constitutive expression of c-REL in uveal melanoma patients and its prognostic significance. METHODS: Detection of c-REL expression was carried out by immunohistochemistry in all 75 patients, and qRT-PCR performed on 58 fresh cases of uveal melanoma along with IL-6 status. Immunoblot was performed to validate immunohistochemistry results. Expression of c-REL protein correlated with clinicopathological parameters and overall survival of patients. RESULTS: Immunohistochemistry results revealed nuclear expression of the c-REL protein (56%) in our cases. Out of 75 cases, 31 cases showed nuclear expression, and 11 cases had cytoplasmic expression. qRT-PCR showed upregulation of the REL gene in 56.89% cases at the transcriptional level. There was a statistically significant difference in the overall survival of patients with c-REL nuclear immunopositivity (p = 0.0048). On multivariate analysis, scleral invasion and c-REL nuclear expression found to be an independent prognostic factor (p < 0.05) CONCLUSIONS: To the best of our knowledge, this was the first study reporting the expression of the c-REL protein in uveal melanoma. Strong nuclear immunoexpression of c-Rel suggests NFκB pathway activation which might be involved in the progression of the disease. Differential expression of c-REL protein may be used as an attractive target for the development of anticancer strategies.

Generation of Foxp3+CD25- Regulatory T-Cell Precursors Requires c-Rel and IκBNS.

Next to the classical developmental route, in which first CD25 and subsequently Foxp3 are induced to generate thymic regulatory T (Treg) cells, an alternative route has been described. This alternative route is characterized by reciprocal induction of Foxp3 and CD25, with CD25 induction being required to rescue developing Treg cells from Foxp3-induced apoptosis. NF-κB has been demonstrated to be crucial for the development of thymic Treg cells via the classical route. However, its impact on the alternative route is poorly characterized. Using single and double deficient mice for key regulators of the classical route, c-Rel and IκBNS, we here demonstrate that NF-κB is essential for the generation of alternative CD25-Foxp3+ precursors, as well. Thus, c-Rel and IκBNS govern both routes of thymic Treg cell development.