ABSTRACT
NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/ß/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.
Subject(s)
Amino Acid Sequence , Gene Expression Regulation , Immunity, Innate , Lipopolysaccharides , Phylogeny , Poly I-C , Sequence Alignment , Strongylocentrotus , Animals , Immunity, Innate/genetics , Poly I-C/pharmacology , Lipopolysaccharides/pharmacology , Strongylocentrotus/genetics , Strongylocentrotus/immunology , Sequence Alignment/veterinary , Gene Expression Regulation/immunology , Cloning, Molecular , Gene Expression Profiling/veterinary , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , NF-kappa B/immunology , Base Sequence , Proto-Oncogene Proteins c-rel/genetics , Proto-Oncogene Proteins c-rel/metabolism , HEK293 CellsABSTRACT
Antibody responses require the proliferative expansion of B cells controlled by affinity-dependent signals. Yet, proliferative bursts are heterogeneous, varying between 0 and 8 divisions in response to the same stimulus. NFκB cRel is activated in response to immune stimulation in B cells and is genetically required for proliferation. Here, we asked whether proliferative heterogeneity is controlled by natural variations in cRel abundance. We developed a fluorescent reporter mTFP1-cRel for the direct observation of cRel in live proliferating B cells. We found that cRel is heterogeneously distributed among naïve B cells, which are enriched for high expressors in a heavy-tailed distribution. We found that high cRel expressors show faster activation of the proliferative program, but do not sustain it well, with population expansion decaying earlier. With a mathematical model of the molecular network, we showed that cRel heterogeneity arises from balancing positive feedback by autoregulation and negative feedback by its inhibitor IκBε, confirmed by mouse knockouts. Using live-cell fluorescence microscopy, we showed that increased cRel primes B cells for early proliferation via higher basal expression of the cell cycle driver cMyc. However, peak cMyc induction amplitude is constrained by incoherent feedforward regulation, decoding the fold change of cRel activity to terminate the proliferative burst. This results in a complex nonlinear, nonmonotonic relationship between cRel expression and the extent of proliferation. These findings emphasize the importance of direct observational studies to complement gene knockout results and to learn about quantitative relationships between biological processes and their key regulators in the context of natural variations.
Subject(s)
B-Lymphocytes , Cell Proliferation , NF-kappa B , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Mice , NF-kappa B/metabolism , Mice, Knockout , Mice, Inbred C57BL , Proto-Oncogene Proteins c-rel/metabolism , Proto-Oncogene Proteins c-rel/geneticsABSTRACT
The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.
Subject(s)
Multiple Sclerosis , Neoplasms , Animals , Mice , CD4-Positive T-Lymphocytes , NF-kappa B , Signal Transduction , Tumor Microenvironment , Proto-Oncogene Proteins c-rel/metabolismABSTRACT
Individual cell sensing of external cues has evolved through the temporal patterns in signaling. Since nuclear factor κB (NF-κB) signaling dynamics have been examined using a single subunit, RelA, it remains unclear whether more information might be transmitted via other subunits. Using NF-κB double-knockin reporter mice, we monitored both canonical NF-κB subunits, RelA and c-Rel, simultaneously in single macrophages by quantitative live-cell imaging. We show that signaling features of RelA and c-Rel convey more information about the stimuli than those of either subunit alone. Machine learning is used to predict the ligand identity accurately based on RelA and c-Rel signaling features without considering the co-activated factors. Ligand discrimination is achieved through selective non-redundancy of RelA and c-Rel signaling dynamics, as well as their temporal coordination. These results suggest a potential role of c-Rel in fine-tuning immune responses and highlight the need for approaches that will elucidate the mechanisms regulating NF-κB subunit specificity.
Subject(s)
NF-kappa B , Proto-Oncogene Proteins c-rel , Mice , Animals , NF-kappa B/metabolism , Ligands , Proto-Oncogene Proteins c-rel/metabolism , Transcription Factor RelA/metabolism , Signal Transduction , Macrophages/metabolismABSTRACT
Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.
Subject(s)
Inflammation , Interleukin-10 , Sphingolipids , Animals , Humans , Mice , Ceramides/chemistry , Ceramides/metabolism , Fatty Acids, Unsaturated/biosynthesis , Fatty Acids, Unsaturated/metabolism , Homeostasis , Immunity, Innate , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Interleukin-10/deficiency , Interleukin-10/genetics , Interleukin-10/metabolism , Proto-Oncogene Proteins c-rel , Sphingolipids/metabolismABSTRACT
Although Parkinson's disease (PD) key neuropathological hallmarks are well known, the underlying pathogenic mechanisms of the disease still need to be elucidated to identify innovative disease-modifying drugs and specific biomarkers. NF-κB transcription factors are involved in regulating several processes associated with neurodegeneration, such as neuroinflammation and cell death, that could be related to PD pathology. NF-κB/c-Rel deficient (c-rel-/-) mice develop a progressive PD-like phenotype. The c-rel-/- mice present both prodromal and motor symptoms as well as key neuropathological features, including nigrostriatal dopaminergic neurons degeneration, accumulation of pro-apoptotic NF-κB/RelA acetylated at the lysine 310 residue (Ac-RelA(lys310)) and progressive caudo-rostral brain deposition of alpha-synuclein. c-Rel inhibition can exacerbate MPTP-induced neurotoxicity in mice. These findings support the claim that misregulation of c-Rel protein may be implicated in PD pathophysiology. In this study, we aimed at evaluating c-Rel levels and DNA-binding activity in human brains and peripheral blood mononuclear cells (PBMCs) of sporadic PD patients. We analyzed c-Rel protein content and activity in frozen substantia nigra (SN) samples from post-mortem brains of 10 PD patients and 9 age-matched controls as well as in PBMCs from 72 PD patients and 40 age-matched controls. c-Rel DNA-binding was significantly lower and inversely correlated with Ac-RelA(lys310) content in post-mortem SN of sporadic PD cases, when compared to healthy controls. c-Rel DNA-binding activity was also reduced in PBMCs of followed-up PD subjects. The decrease of c-Rel activity in PBMCs from PD patients appeared to be independent from dopaminergic medication or disease progression, as it was evident even in early stage, drug-naïve patients. Remarkably, the levels of c-Rel protein were comparable in PD and control subjects, pointing out a putative role for post-translational modifications of the protein in c-Rel dysfunctions. These findings support that PD is characterized by the loss of NF-κB/c-Rel activity that potentially has a role in PD pathophysiology. Future studies will be aimed at addressing whether the reduction of c-Rel DNA-binding could constitute a novel biomarker for PD.
Subject(s)
MPTP Poisoning , Parkinson Disease , Humans , Mice , Animals , NF-kappa B/metabolism , Parkinson Disease/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Leukocytes, Mononuclear/metabolism , Substantia Nigra/metabolism , Dopaminergic Neurons/metabolism , MPTP Poisoning/pathologyABSTRACT
BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC. APPROACH AND RESULTS: Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone. CONCLUSION: Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Humans , Mice , Carcinogenesis/genetics , Carcinoma, Hepatocellular/pathology , DNA Damage , Doxorubicin/pharmacology , Hepatocytes/metabolism , Liver Neoplasms/metabolism , Mice, Knockout , NF-kappa B/metabolism , Proto-Oncogene Proteins c-rel/metabolismABSTRACT
Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-κB subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.
Subject(s)
Mutation, Missense , NF-kappa B , DNA , HEK293 Cells , Humans , NF-kappa B/metabolism , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-rel/metabolismABSTRACT
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.
Subject(s)
Mycobacterium bovis , Mycobacterium tuberculosis , Proto-Oncogene Proteins c-rel/genetics , Tuberculosis , Adult , BCG Vaccine , Basic Helix-Loop-Helix Transcription Factors , Child , Homeodomain Proteins , Humans , Interleukin-10/genetics , Interleukin-12/genetics , Tuberculosis/geneticsABSTRACT
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.
Subject(s)
Ataxin-1/genetics , Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Leukemia-Lymphoma, Adult T-Cell/pathology , Mutation , Proto-Oncogene Proteins c-rel/genetics , Repressor Proteins/genetics , Animals , DNA Copy Number Variations , Female , Genome, Human , Humans , Leukemia-Lymphoma, Adult T-Cell/genetics , Mice , Mice, Inbred C57BL , Prognosis , Survival Rate , Exome SequencingABSTRACT
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.
Subject(s)
Immunity, Innate , Lung/immunology , Lymphocyte Subsets/immunology , Proto-Oncogene Proteins c-rel/immunology , Animals , Asthma/immunology , Asthma/pathology , Disease Models, Animal , Female , Gene Expression , In Vitro Techniques , Interleukin-33/immunology , Lung/pathology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Proto-Oncogene Proteins c-rel/deficiency , Proto-Oncogene Proteins c-rel/geneticsABSTRACT
Type I interferon (IFN-I) is a key component of the host innate immune system. To establish efficient replication, viruses have developed several strategies to escape from the host IFN response. Japanese encephalitis virus (JEV) NS1', a larger NS1-related protein, is known to inhibit the mitochondrial antiviral signaling (MAVS)-mediated IFN-ß induction by increasing the binding of transcription factors (CREB and c-Rel) to the microRNA 22 (miRNA-22) promoter. However, the mechanism by which NS1' induces the recruitment of CREB and c-Rel onto the miRNA-22 promoter is unknown. Here, we found that JEV NS1' protein interacts with the host cyclin-dependent kinase 1 (CDK1) protein. Mechanistically, NS1' interrupts the CDC25C phosphatase-mediated dephosphorylation of CDK1, which prolongs the phosphorylation status of CDK1 and leads to the inhibition of MAVS-mediated IFN-ß induction. Furthermore, the CREB phosphorylation and c-Rel activation through the IκBα phosphorylation were observed to be enhanced upon the augmentation of CDK1 phosphorylation by NS1'. The abrogation of CDK1 activity by a small-molecule inhibitor significantly suppressed the JEV replication in vitro and in vivo. Moreover, the administration of CDK1 inhibitor protected the wild-type mice from JEV-induced lethality but showed no effect on the MAVS-/- mice challenged with JEV. In conclusion, our study provides new insight into the mechanism of JEV immune evasion, which may lead to the development of novel therapeutic options to treat JEV infection. IMPORTANCE Japanese encephalitis virus (JEV) is the main cause of acute human encephalitis in Asia. The unavailability of specific treatment for Japanese encephalitis demands a better understanding of the basic cellular mechanisms that contribute to the onset of disease. The present study identifies a novel interaction between the JEV NS1' protein and the cellular CDK1 protein, which facilitates the JEV replication by dampening the cellular antiviral response. This study sheds light on a novel mechanism of JEV replication, and thus our findings could be employed for developing new therapies against JEV infection.
Subject(s)
CDC2 Protein Kinase/metabolism , Encephalitis Virus, Japanese/immunology , Immune Evasion/immunology , Interferon-beta/immunology , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology , Animals , CDC2 Protein Kinase/antagonists & inhibitors , CREB-Binding Protein/metabolism , Cell Line, Tumor , Cricetinae , Encephalitis, Japanese/immunology , HeLa Cells , Humans , Immunity, Innate/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , NF-KappaB Inhibitor alpha/metabolism , Phosphorylation/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-rel/metabolism , cdc25 Phosphatases/metabolismABSTRACT
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.
Subject(s)
Genes, rel , Primary Immunodeficiency Diseases/genetics , Primary Immunodeficiency Diseases/immunology , Proto-Oncogene Proteins c-rel/deficiency , Proto-Oncogene Proteins c-rel/genetics , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Child , Consanguinity , Female , Hematopoietic Stem Cell Transplantation , Homozygote , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Lymphocyte Activation , Lymphocytes/classification , Lymphocytes/immunology , Mutation , Myeloid Cells/immunology , Primary Immunodeficiency Diseases/therapy , Protein IsoformsABSTRACT
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.
Subject(s)
Encephalitis, Herpes Simplex/metabolism , Interferon Type I/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Animals , Encephalitis, Herpes Simplex/virology , Female , Male , Mice , Mice, Inbred C57BL , Mutation , Proto-Oncogene Proteins c-rel/genetics , Signal Transduction , Simplexvirus/genetics , Simplexvirus/pathogenicity , Simplexvirus/physiology , T-Lymphocytes/metabolism , T-Lymphocytes/virologyABSTRACT
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.
Subject(s)
Immunity, Innate/drug effects , Interleukin-33/pharmacology , Lung/drug effects , Lymphocyte Activation/drug effects , Lymphocytes/drug effects , Pneumonia/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Animals , Bone Marrow/drug effects , Bone Marrow/immunology , Bone Marrow/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Interleukin-13/metabolism , Interleukin-5/metabolism , Lung/immunology , Lung/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/metabolism , Papain , Pneumonia/chemically induced , Pneumonia/genetics , Pneumonia/immunology , Proto-Oncogene Proteins c-rel/geneticsSubject(s)
Integrases/genetics , Paternal Inheritance , Recombination, Genetic , Transcription Factors/genetics , Animals , Female , Genotype , Humans , Integrases/metabolism , Male , Mice, Transgenic , Mutation , Phenotype , Promoter Regions, Genetic , Proto-Oncogene Proteins c-rel/genetics , Proto-Oncogene Proteins c-rel/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Transcription Factor RelB/genetics , Transcription Factor RelB/metabolismABSTRACT
Target evaluation and rational drug design rely on identifying and characterising small-molecule binding sites on therapeutically relevant target proteins. Immunotherapeutics development is especially challenging because of complex disease etiology and heterogenous nature of targets. c-Rel protein, a promising target in many human inflammatory and cancer pathologies, was selected as a case study for an effective in silico screening platform development since this transcription factor currently has no successful therapeutic inhibitors or modulators. This study introduces a novel in silico screening approach to probe binding sites using structural validation sets, molecular modelling and describes a method of a computer-aided drug design when a crystal structure is not available for the target of interest. In addition, we showed that binding sites can be analysed with the machine learning as well as molecular simulation approaches to help assess and systematically analyse how drug candidates can exert their mode of action. Finally, this cutting-edge approach was subjected to a high through-put virtual screen of selected 34 M drug-like compounds filtered from a library of 659 M compounds by identifying the most promising structures and proposing potential action mechanisms for the future development of highly selective human c-Rel inhibitors and/or modulators.
Subject(s)
Proto-Oncogene Proteins c-rel , Drug Discovery , Ligands , Molecular Docking Simulation , Protein CABSTRACT
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.
Subject(s)
Cytotoxicity, Immunologic , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Animals , Cells, Cultured , Cytokines/biosynthesis , Cytotoxicity, Immunologic/genetics , Granzymes/metabolism , Humans , Melanoma, Experimental , Mice , Mice, Knockout , Models, Biological , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism , Proto-Oncogene Proteins c-rel/geneticsABSTRACT
Sorting nexin 10 (SNX10) has been reported as a critical regulator in macrophage function, and germline SNX10 knockout effectively alleviated mouse colitis. Here, we investigated the precise role of SNX10 in inflammatory responses in macrophages in mouse colitis, and explored the druggability of SNX10 as a therapeutic target for inflammatory bowel disease (IBD). Our results revealed that myeloid-specific SNX10 deletion alleviated inflammation and pathological damage induced by dextran sulfate sodium (DSS). In vitro experiments showed that SNX10 deletion contributed to inflammation elimination by inhibiting PIKfyve-mediated TANK-binding kinase 1 (TBK1) /c-Rel signaling activation. Further study provided rational mechanism that SNX10 was required for the recruitment of PIKfyve to the TRIF-positive endosomes, through which PIKfyve activated TBK1/c-Rel for LPS-induced inflammation response. Based on the structure of SNX10, we discovered a new small-molecule inhibitor DC-SX029, which targeted SNX10 to block the SNX10-PIKfyve interaction, thereby decreased the TBK1/c-Rel signaling activation. Additionally, therapeutic efficiency of DC-SX029 was evaluated in both DSS-induced and IL10-deficient mouse colitis models. Our data demonstrate a new mechanism by which SNX10-PIKfyve interaction regulates LPS-induced inflammation response in macrophages via the TBK1/c-Rel signaling pathway. In vivo and in vitro pharmacological studies of SNX10 protein-protein interaction (PPI) inhibitor DC-SX029 demonstrate the feasibility of targeting SNX10 in IBD treatment.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Colitis/drug therapy , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Signal Transduction/drug effects , Sorting Nexins/drug effects , Animals , Colitis/metabolism , Disease Models, Animal , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sorting Nexins/metabolismABSTRACT
Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype characterized by poor clinical outcome. In recent years, numerous advancements have been made to better understand the biological landscape of TNBC, though appropriate targets still remain to be determined. In the present study, we have determined that the expression levels of FGF2 and S100A4 are higher in TNBC with respect to non-TNBC patients when analyzing "The Invasive Breast Cancer Cohort of The Cancer Genome Atlas" (TCGA) dataset. In addition, we have found that the gene expression of FGF2 is positively correlated with S100A4 in TNBC samples. Performing quantitative PCR, Western blot, CRISPR/Cas9 genome editing, promoter studies, immunofluorescence analysis, subcellular fractionation studies, and ChIP assays, we have also demonstrated that FGF2 induces in TNBC cells the upregulation and secretion of S100A4 via FGFR1, along with the ERK1/2-AKT-c-Rel transduction signaling. Using conditioned medium from TNBC cells stimulated with FGF2, we have also ascertained that the paracrine activation of the S100A4/RAGE pathway triggers angiogenic effects in vascular endothelial cells (HUVECs) and promotes the migration of cancer-associated fibroblasts (CAFs). Collectively, our data provide novel insights into the action of the FGF2/FGFR1 axis through S100A4 toward stimulatory effects elicited in TNBC cells.