RESUMO
OBJECTIVES: While the gastrointestinal symptom cluster (GISC) is common in patients receiving chemotherapy, limited information is available on its underlying mechanism(s). Emerging evidence suggests a role for inflammatory processes through the actions of the nuclear factor kappa B (NF-κB) signaling pathway. This study evaluated for associations between a GISC and levels of DNA methylation for genes within this pathway. METHODS: Prior to their second or third cycle of chemotherapy, 1071 outpatients reported symptom occurrence using the Memorial Symptom Assessment Scale. A GISC was identified using exploratory factor analysis. Differential methylation analyses were performed in two independent samples using EPIC (n = 925) and 450K (n = 146) microarrays. Trans expression-associated CpG (eCpG) loci for 56 NF-κB signaling pathway genes were evaluated. Loci significance were assessed using an exploratory false discovery rate (FDR) of 25% for the EPIC sample. For the validation assessment using the 450K sample, significance was assessed at an unadjusted p-value of 0.05. RESULTS: For the EPIC sample, the GISC was associated with increased expression of lymphotoxin beta (LTB) at one differentially methylated trans eCpG locus (cg03171795; FDR = 0.168). This association was not validated in the 450K sample. CONCLUSIONS: This study is the first to identify an association between a GISC and epigenetic regulation of a gene that is involved in the initiation of gastrointestinal immune responses. Findings suggest that increased LTB expression by hypermethylation of a trans eCpG locus is involved in the occurrence of this cluster in patients receiving chemotherapy. LTB may be a potential therapeutic target for this common cluster.
Assuntos
Epigênese Genética , Neoplasias , Humanos , Linfotoxina-beta , Síndrome , NF-kappa B , Metilação de DNA , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Tumor-associated tertiary lymphoid structures are ectopic lymphoid aggregates that have considerable morphological, cellular, and molecular similarity to secondary lymphoid organs, particularly lymph nodes. Tumor vessels expressing peripheral node addressin (PNAd) are hallmark features of these structures. Previous work from our laboratory demonstrated that PNAd is displayed on intratumoral vasculature of murine tumors, and its expression is controlled by the engagement of lymphotoxin-α3, secreted by effector CD8 T cells, with tumor necrosis factor receptors (TNFR) on tumor endothelial cells (TEC). The goals of the present work were: 1) to identify differences in expression of genes encoding the scaffolding proteins and glycosyl transferases associated with PNAd biosynthesis in TEC and lymph node blood endothelial cells (LN BEC); and 2) to determine which of these PNAd associated components are regulated by TNFR signaling. We found that the same genes encoding scaffolding proteins and glycosyl transferases were upregulated in PNAd+ LN BEC and PNAd+ TEC relative to their PNAdneg counterparts. The lower level of PNAd expression on TEC vs LN BEC was associated with relatively lower expression of these genes, particularly the carbohydrate sulfotransferase Chst4. Loss of PNAd on TEC in the absence of TNFR signaling was associated with lack of upregulation of these same genes. A small subset of PNAd+ TEC remaining in the absence of TNFR signaling showed normal upregulation of a subset of these genes, but reduced upregulation of genes encoding the scaffolding proteins podocalyxin and nepmucin, and carbohydrate sulfotransferase Chst2. Lastly, we found that checkpoint immunotherapy augmented both the fraction of TEC expressing PNAd and their surface level of this ligand. This work points to strong similarities in the regulation of PNAd expression on TEC by TNFR signaling and on LN BEC by lymphotoxin-ß receptor signaling, and provides a platform for the development of novel strategies that manipulate PNAd expression on tumor vasculature as an element of cancer immunotherapy.
Assuntos
Células Endoteliais , Neoplasias , Animais , Ligantes , Linfotoxina-alfa/fisiologia , Linfotoxina-beta , Camundongos , Neoplasias/genética , Receptores do Fator de Necrose Tumoral , SulfotransferasesRESUMO
Cutaneous melanoma (CM) is the most common skin cancer and one of the most aggressive cancers and its incidence has risen dramatically over the past few decades. The tumor microenvironment (TME) plays a crucial role in the occurrence and development of cutaneous melanoma. Nevertheless, the dynamics modulation of the immune and stromal components in the TME is not fully understood. In this study, 471 CM samples were obtained from TCGA database, and the ratio of tumor-infiltrating immune cells (TICs) in the TME were estimated using the ESTIMATE algorithms and CIBERSORT computational method. The differently expressed genes (DEGs) were applied to GO and KEGG function enrichment analysis, establishment of protein-protein interaction (PPI) network and univariate Cox regression analysis. Subsequently, we identified a predictive factor: HLA-DRB1 (major histocompatibility complex, class II, DR beta 1) by the intersection analysis of the hub genes of PPI network and the genes associated with the prognosis of the CM patients obtained by univariate Cox regression analysis. Correlation analysis and survival analysis showed that the expression level of HLA-DRB1 was negatively correlated with the Stage of the patients while positively correlated with the survival, prognosis and TME of melanoma. The GEPIA web server and the representative immunohistochemical images of HLA-DRB1 in the normal skin tissue and melanoma tissue from the Human Protein Atlas (HPA) database were applied to validate the expression level of HLA-DRB1. CIBERSORT analysis for the ratio of TICs indicated that 9 types of TICs were positively correlated with the expression level of HLA-DRB1 and only 4 types of TICs were negatively correlated with the expression level of HLA-DRB1. These results suggested that the expression level of HLA-DRB1 may be related to the immune activity of the TME and may affect the prognosis of CM patients by changing the status of the TME.
Assuntos
Melanoma , Neoplasias Cutâneas , Regulação Neoplásica da Expressão Gênica , Cadeias HLA-DRB1/genética , Humanos , Linfotoxina-beta/genética , Melanoma/genética , Prognóstico , Fator Regulador X1/genética , Neoplasias Cutâneas/genética , Microambiente Tumoral/genética , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is highly recurrent. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, promote malignancy; however, the mechanisms underlying their actions are obscure. We aimed to identify CAF-specific proteins in HCC and determine whether they could be potential therapeutic targets. METHODS: Using comprehensive proteomic analysis of CAFs and noncancerous fibroblasts (NFs) primary-cultured from resected HCC specimens from the same patients, CAF-specific proteins were identified. Immunohistochemistry for versican (VCAN) was performed on cancerous tissues obtained from 239 patients with HCC. Conditioned medium from CAFs transfected with siRNA for VCAN was analyzed in vitro. RESULTS: CAFs significantly promoted HCC cell proliferation, migration, and invasion (p < 0.01, 0.01, and 0.01, respectively) compared with NFs. VCAN was upregulated in CAFs, and its stromal level correlated with poor differentiation (p = 0.009) and positive vascular invasion (p = 0.003). Stromal VCAN level was also associated with significantly lower overall (p = 0.002) and relapse-free (p < 0.001) survival rates. It also independently predicted prognosis and recurrence. VCAN-knockdown CAFs significantly suppressed HCC cell migration and invasion compared with negative control. CONCLUSIONS: VCAN secreted from CAFs promoted malignant transformation of HCC cells and has potential as a new therapeutic target in HCC.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Hepatocelular , Neoplasias Hepáticas , Fibroblastos Associados a Câncer/patologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Neoplasias Hepáticas/patologia , Linfotoxina-beta/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico , Proteômica , RNA Interferente Pequeno , Microambiente Tumoral , Versicanas/metabolismoRESUMO
Regulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αß and LEC LTß receptor (LTßR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTßR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.
Assuntos
Células Endoteliais , Linfócitos T Reguladores , Adenosina , Animais , Fatores de Transcrição Forkhead/genética , Linfotoxina-beta , Camundongos , NF-kappa BRESUMO
Lymphotoxin-ß-receptor deficient (LTßR-/-) and Tumor Necrosis Factor Receptor p55 deficient (TNFRp55-/-) mice show defects in liver regeneration (LR) after partial hepatectomy (PHx) with significantly increased mortality. LTßR and TNFRp55 belong to the core members of the TNF/TNFR superfamily. Interestingly, combined failure of LTßR and TNFRp55 signaling after PHx leads to a complete defect in LR. Here, we first addressed the question which liver cell population crucially requires LTßR signaling for efficient LR. To this end, mice with a conditionally targeted LTßR allele (LTßRfl/fl) were crossed to AlbuminCre and LysozymeMCre mouse lines to unravel the function of the LTßR on hepatocytes and monocytes/macrophages/Kupffer cells, respectively. Analysis of these mouse lines clearly reveals that LTßR is required on hepatocytes for efficient LR while no deficit in LR was found in LTßRfl/fl × LysMCre mice. Second, the molecular basis for the cooperating role of LTßR and TNFRp55 signaling pathways in LR was investigated by transcriptome analysis of etanercept treated LTßR-/- (LTßR-/-/ET) mice. Bioinformatic analysis and subsequent verification by qRT-PCR identified novel target genes (Cyclin-L2, Fas-Binding factor 1, interferon-related developmental regulator 1, Leucyl-tRNA Synthetase 2, and galectin-4) that are upregulated by LTßR/TNFRp55 signaling after PHx and fail to be upregulated after PHx in LTßR-/-/ET mice.
Assuntos
Regeneração Hepática , Animais , Hepatectomia , Hepatócitos , Linfotoxina-beta , Transdução de SinaisRESUMO
Infection of the mammalian host with African trypanosomes begins when the tsetse fly vector injects the parasites into the skin dermis during blood feeding. After injection into the skin, trypanosomes first accumulate in the draining lymph node before disseminating systemically. Whether this early accumulation within the draining lymph node is important for the trypanosomes to establish infection was not known. Lymphotoxin-ß-deficient mice (LTß-/- mice) lack most secondary lymphoid tissues, but retain the spleen and mesenteric lymph nodes. These mice were used to test the hypothesis that the establishment of infection after intradermal (ID) T. brucei infection would be impeded in the absence of the skin draining lymph nodes. However, LTß-/- mice revealed greater susceptibility to ID T. brucei infection than wild-type mice, indicating that the early accumulation of the trypanosomes in the draining lymph nodes was not essential to establish systemic infection. Although LTß-/- mice were able to control the first parasitemia wave as effectively as wild-type mice, they were unable to control subsequent parasitemia waves. LTß-/- mice also lack organized B cell follicles and germinal centers within their remaining secondary lymphoid tissues. As a consequence, LTß-/- mice have impaired immunoglobulin (Ig) isotype class-switching responses. When the disturbed microarchitecture of the B cell follicles in the spleens of LTß-/- mice was restored by reconstitution with wild-type bone marrow, their susceptibility to ID T. brucei infection was similar to that of wild-type control mice. This effect coincided with the ability to produce significant serum levels of Ig isotype class-switched parasite-specific antibodies. Thus, our data suggest that organized splenic microarchitecture and the production of parasite-specific Ig isotype class-switched antibodies are essential for the control of ID African trypanosome infections.
Assuntos
Linfonodos/imunologia , Pele/parasitologia , Baço/imunologia , Tripanossomíase Africana/imunologia , Animais , Anticorpos Antiprotozoários , Feminino , Linfotoxina-beta/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/imunologia , Baço/parasitologia , Trypanosoma brucei bruceiRESUMO
Mycophenolic acid (MPA) is an immunosuppressant commonly used to prevent renal transplant rejection and treat glomerulonephritis. MPA inhibits IMPDH2 within stimulated lymphocytes, reducing guanosine synthesis. Despite the widespread use of MPA, interindividual variability in response remains with rates of allograft rejection up to 15% and approximately half of individuals fail to achieve complete remission to lupus nephritis. We sought to identify contributors to interindividual variability in MPA response, hypothesizing that the HPRT1 salvage guanosine synthesis contributes to variability. MPA sensitivity was measured in 40 healthy individuals using an ex vivo lymphocyte viability assay. Measurement of candidate gene expression (n ± 40) and single-cell RNA-sequencing (n ± 6) in lymphocytes was performed at baseline, poststimulation, and post-MPA treatment. After stimulation, HPRT1 expression was 2.1-fold higher in resistant individuals compared with sensitive individuals (P ± 0.049). Knockdown of HPRT1 increased MPA sensitivity (12%; P ± 0.003), consistent with higher expression levels in resistant individuals. Sensitive individuals had higher IMPDH2 expression and 132% greater stimulation. In lymphocyte subpopulations, differentially expressed genes between sensitive and resistant individuals included KLF2 and LTB. Knockdown of KLF2 and LTB aligned with the predicted direction of effect on proliferation. In sensitive individuals, more frequent receptor-ligand interactions were observed after stimulation (P ± 0.0004), but fewer interactions remained after MPA treatment (P ± 0.0014). These data identify a polygenic transcriptomic signature in lymphocyte subpopulations predictive of MPA response. The degree of lymphocyte stimulation, HPRT1, KLF2, and LTB expression may serve as markers of MPA efficacy.
Assuntos
Imunossupressores/farmacologia , Ativação Linfocitária/genética , Linfócitos/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Adulto , Idoso , Variação Biológica da População/imunologia , Biomarcadores Farmacológicos , Resistência a Medicamentos/genética , Feminino , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Voluntários Saudáveis , Humanos , Hipoxantina Fosforribosiltransferase/genética , Imunossupressores/uso terapêutico , Transplante de Rim/efeitos adversos , Fatores de Transcrição Kruppel-Like/genética , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Linfotoxina-beta/genética , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/uso terapêutico , Cultura Primária de Células , RNA-Seq , Análise de Célula Única , Adulto JovemRESUMO
It has been established that inflammation plays an important role in bone formation and bone loss. Although a lot is known about the role of TNF-α in bone health, very little is understood about TNF-ß, also called lymphotoxin. In this report, we examine the effect of TNF-ß on osteogenic differentiation of mesenchymal stem cells (MSCs) and its modulation by resveratrol. Monolayer and high-density cultures of MSCs were treated with osteogenic induction medium with/without TNF-ß, Sirt1 inhibitor nicotinamide (NAM), antisense oligonucleotides against Sirt1 (ASO) and/or Sirt1 stimulator resveratrol. We found that TNF-ß inhibits, in a similar way to NAM or Sirt1-ASO, the early stage of osteogenic differentiation of MSCs and this was accompanied with downregulation of bone-specific matrix, ß1-integrin, Runx2 and with upregulation of NF-κB phosphorylation and NF-κB-regulated gene products involved in the inflammatory, degradative processes and apoptosis. However, resveratrol reversed TNF-ß- and NAM-suppressed MSCs osteogenesis by activation of Sirt1 and Runx2 that led to osteoblast differentiation. Furthermore, downregulation of Sirt1 by mRNA inhibited the effect of resveratrol, highlighting the important impact of this enzyme in the TNF-ß signaling pathway. Finally, resveratrol was able to manifest its effect both by suppression of TNF-ß-induced NF-κB and through direct activation of the Sirt1 and Runx2 pathway. Thus, through these studies, we present a mechanism by which a T cell-derived cytokine, TNF-ß can affect bone formation through modulation of MSCs differentiation that involves NF-κB, Sirt1, Runx2 and resveratrol reversed TNF-ß-promoted impairments in MSCs osteogenesis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Linfotoxina-beta/farmacologia , Células-Tronco Mesenquimais/citologia , Osteoblastos , Osteogênese , Resveratrol/farmacologia , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cães , NF-kappa B/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Sirtuína 1/metabolismoRESUMO
This commentary highlights the article by Francipane et al that studied the molecular signals supporting kidney vascularization in host lymphoid sites and omenta.
Assuntos
Receptor beta de Linfotoxina , Linfotoxina-alfa , Animais , Linfotoxina-beta , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Head and neck squamous cell carcinomas (HNSCC) promote inflammation in the tumor microenvironment through aberrant NF-κB activation, but the genomic alterations and pathway networks that modulate NF-κB signaling have not been fully dissected. Here, we analyzed genome and transcriptome alterations of 279 HNSCC specimens from The Cancer Genome Atlas (TCGA) cohort and identified 61 genes involved in NF-κB and inflammatory pathways. The top 30 altered genes were distributed across 96% of HNSCC samples, and their expression was often correlated with genomic copy-number alterations (CNA). Ten of the amplified genes were associated with human papilloma virus (HPV) status. We sequenced 15 HPV- and 11 HPV+ human HNSCC cell lines, and three oral mucosa keratinocyte lines, and supervised clustering revealed that 28 of 61 genes exhibit altered expression patterns concordant with HNSCC tissues and distinct signatures related to their HPV status. RNAi screening using an NF-κB reporter line identified 16 genes that are induced by TNFα or Lymphotoxin-ß (LTß) and implicated in the classic and/or alternative NF-κB pathways. Knockdown of TNFR, LTBR, or selected downstream signaling components established cross-talk between the classic and alternative NF-κB pathways. TNFα and LTß induced differential gene expression involving the NF-κB, IFNγ, and STAT pathways, inflammatory cytokines, and metastasis-related genes. Improved survival was observed in HNSCC patients with elevated gene expression in T-cell activation, immune checkpoints, and IFNγ and STAT pathways. These gene signatures of NF-κB activation, which modulate inflammation and responses to the immune therapy, could serve as potential biomarkers in future clinical trials.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , NF-kappa B/metabolismo , Transdução de Sinais/genética , Linhagem Celular , Proliferação de Células , Variações do Número de Cópias de DNA , Bases de Dados Genéticas , Feminino , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Inflamação , Interferon gama/metabolismo , Linfotoxina-beta/metabolismo , Masculino , NF-kappa B/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Fatores de Transcrição STAT/metabolismo , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismoRESUMO
IL-4 is critical for differentiation of Th2 cells and antibody isotype switching, but our work demonstrated that it is produced in the peripheral LN under both Type 2, and Type 1 conditions, raising the possibility of other functions. We found that IL-4 is vital for proper positioning of hematopoietic and stromal cells in steady state, and the lack of IL-4 or IL-4Rα correlates with disarrangement of both follicular dendritic cells and CD31+ endothelial cells. We observed a marked disorganization of B cells in these mice, suggesting that the lymphocyte-stromal cell axis is maintained by the IL-4 signaling pathway. This study showed that absence of IL-4 correlates with significant downregulation of Lymphotoxin alpha (LTα) and Lymphotoxin beta (LTß), critical lymphokines for the development and maintenance of lymphoid organs. Moreover, immunization of IL-4 deficient mice with Type 2 antigens failed to induce lymphotoxin production, LN reorganization, or germinal center formation, while this process is IL-4 independent following Type 1 immunization. Additionally, we found that Type 1 antigen mediated LN reorganization is dependent on IFN-γ in the absence of IL-4. Our findings reveal a role of IL-4 in the maintenance of peripheral lymphoid organ microenvironments during homeostasis and antigenic challenge.
Assuntos
Proliferação de Células , Interleucina-4/imunologia , Receptores de Superfície Celular/imunologia , Células Estromais/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Linfotoxina-alfa/imunologia , Linfotoxina-alfa/metabolismo , Linfotoxina-beta/imunologia , Linfotoxina-beta/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-ß (LTß), receptor LTßR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTßR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTß, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTßR, NIK, and RELB proteins. Recombinant LTß, and siRNA depletion of endogenous LTßR and NIK, modulated expression of LTßR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTß induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTß-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTß is expressed. Our findings show that LTß/LTßR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.
Assuntos
Carcinoma de Células Escamosas/secundário , Neoplasias de Cabeça e Pescoço/patologia , Receptor beta de Linfotoxina/metabolismo , Linfotoxina-alfa/metabolismo , Linfotoxina-beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição RelB/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Receptor beta de Linfotoxina/genética , Linfotoxina-alfa/genética , Linfotoxina-beta/genética , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fator de Transcrição RelB/genética , Células Tumorais Cultivadas , Quinase Induzida por NF-kappaBRESUMO
BACKGROUND & AIMS: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction. METHODS: We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin ß (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (CyldΔLPC mice and Cyld/RelbΔLPC mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl4 to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB. RESULTS: In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of CyldΔLPC mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/RelbΔLPC mice. Compared with livers from control mice, livers from CyldΔLPC mice (but not Cyld/RelbΔLPC mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl4 exposure. CyldΔLPC mice and Cyld/RelbΔLPC mice had improved liver function on the DDC diet compared with control mice fed the DDC diet. CONCLUSION: Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in CyldΔLPC mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.
Assuntos
Ductos Biliares/metabolismo , Ductos Biliares/patologia , Colangite Esclerosante/metabolismo , Citocinas/genética , Hepatopatias/metabolismo , Fator de Transcrição RelB/metabolismo , Adolescente , Adulto , Idoso , Animais , Tetracloreto de Carbono , Núcleo Celular , Proliferação de Células , Células Cultivadas , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Dicarbetoxi-Di-Hidrocolidina , Células Epiteliais/metabolismo , Feminino , Fibrose , Técnicas de Silenciamento de Genes , Humanos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Receptor beta de Linfotoxina/agonistas , Linfotoxina-beta/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Tecido Parenquimatoso/patologia , Transporte Proteico , Proto-Oncogene Mas , RNA Mensageiro/metabolismo , Fator de Transcrição RelB/genética , Adulto JovemRESUMO
Over 1.5 million individuals in the United States are afflicted with inflammatory bowel disease (IBD). While the progression of IBD is multifactorial, chronic, unresolved inflammation certainly plays a key role. Additionally, while multiple immune mediators have been shown to affect pathogenesis, a comprehensive understanding of disease progression is lacking. Previous work has demonstrated that a member of the TNF superfamily, TNFSF14 (LIGHT), which is pro-inflammatory in several contexts, surprisingly plays an important role in protection from inflammation in mouse models of colitis, with LIGHT deficient mice having more severe disease pathogenesis. However, LIGHT is a single member of a complex signaling network. It signals through multiple receptors, including herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTßR); these two receptors in turn can bind to other ligands. It remains unknown which receptors and competing ligands can mediate or counteract the outcome of LIGHT-signaling during colitis. Here we demonstrate that LIGHT signaling through LTßR, rather than HVEM, plays a critical role in the progression of DSS-induced colitis, as LTßR deficient mice exhibit a more severe disease phenotype. Further, mice deficient in LTαß do not exhibit differential colitis progression compared to WT mice. However, deletion of both LIGHT and LTαß, but not deletion of both LTαß and LTßR, resulted in a reversal of the adverse effects associated with the loss of LIGHT. In sum, the LIGHT/LTαß/LTßR signaling network contributes to DSS colitis, but there may be additional receptors or indirect effects, and therefore, the relationships between these receptors and ligands remains enigmatic.
Assuntos
Colite/imunologia , Doenças Inflamatórias Intestinais/imunologia , Receptor beta de Linfotoxina/metabolismo , Linfotoxina-beta/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Colite/induzido quimicamente , Sulfato de Dextrana , Modelos Animais de Doenças , Progressão da Doença , Humanos , Receptor beta de Linfotoxina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: This study shows the relationship between host factors and environmental factors in the influence of susceptibility to loss of dental implants. PURPOSE: The aim of this study was to investigate the association of clinical aspects and tag SNPs of the genes LTA, TNFA, and LTB with dental implant loss. MATERIALS AND METHODS: The subjects consisted of 244 patients, divided into two groups: control group (C)-163 individuals who did not lose any implants, being in function for at least 6 months; and study group (S)-81 individuals who had lost at least one implant. DNA was collected from saliva, and the genotypes were determined by real time PCR. Univariate and multivariate analysis were employed p < .05. RESULTS: After multivariate analysis, dental implant loss remained associated with the presence of teeth (p = .011), a larger amount of placed implants (p = .001), and allelle C of rs2009658 of the LTA gene (p = .006). For the other tag SNPs of these studied genes, there was no association between the groups C and S with dental implants loss. CONCLUSION: Presence of teeth, number of placed implants and allele C of rs2009658 of LTA gene were associated with implant loss.
Assuntos
Implantes Dentários , Falha de Restauração Dentária , Linfotoxina-alfa/genética , Linfotoxina-beta/genética , Osseointegração/genética , Polimorfismo de Nucleotídeo Único , Fator de Necrose Tumoral alfa/genética , Estudos de Casos e Controles , Implantação Dentária Endóssea , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.
Assuntos
Linfócitos B/imunologia , Catepsinas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Baço/imunologia , Animais , Células CHO , Catepsinas/antagonistas & inibidores , Linhagem Celular , Cricetulus , Receptor beta de Linfotoxina/biossíntese , Linfotoxina-alfa/biossíntese , Linfotoxina-beta/biossíntese , Camundongos , Camundongos Transgênicos , Sirolimo/farmacologia , Baço/citologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
Cytokine production is essential for follicular dendritic cell (FDC) maintenance and organization of germinal centres. In follicular lymphoma, FDCs are often disarrayed and may lack antigens indicative of terminal differentiation. We investigated the in situ distribution of cells producing lymphotoxin-beta (LTB), lymphotoxin-alpha (LTA), and tumour necrosis factor-alpha (TNFA) transcripts in human reactive lymph nodes and in follicular lymphomas with follicular or diffuse growth pattern. LTB was the cytokine most abundantly produced in germinal centres. LTB was present in nearly 90% of germinal centre cells whereas LTA and TNFA were detected in 30 and 50%, respectively. Moreover, the amount of LTB expressed in reactive germinal centre cells was 80-fold higher than that of LTA and 20-fold higher than that of TNFA. LTB-positive cells were more numerous in the germinal centre dark zone, whereas expression of the FDC proteins CD21, CD23, VCAM, and CXCL13 was more intense in the light zone. Tumour cells of follicular lymphomas produced less LTB than reactive germinal centre cells. The results of the in situ study were confirmed by RT-PCR; LTB was significantly more abundant in reactive lymph nodes than in follicular lymphoma, with the lowest values detected in predominantly diffuse follicular lymphoma. In neoplastic follicles, low production of LTB by tumour B cells was associated with weaker expression of CD21+/CD23+ by FDCs. Our findings detail for the first time the distribution of LTA-, LTB-, and TNFA-producing cells in human reactive germinal centres and in follicular lymphoma. They suggest the possibility that impaired tumour-cell LTB production may represent a determinant of FDC phenotype loss and for defective follicular organization in follicular lymphoma.
Assuntos
Linfoma Folicular/metabolismo , Linfotoxina-beta/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Células Dendríticas Foliculares/metabolismo , Feminino , Centro Germinativo/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfonodos/metabolismo , Linfoma Folicular/patologia , Linfotoxina-alfa/genética , Linfotoxina-alfa/metabolismo , Linfotoxina-beta/genética , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
The transcription factor nuclear factor κB (NF-κB) regulates various biological processes, including inflammatory responses. We previously reported that eudesmane-type sesquiterpene lactones inhibited multiple steps in the canonical NF-κB signaling pathway induced by tumor necrosis factor-α and interleukin-1α. In contrast, the biological activities of eudesmane-type sesquiterpene lactones on the non-canonical NF-κB signaling pathway remain unclear. In the present study, we found that (11S)-2α-bromo-3-oxoeudesmano-12,6α-lactone, designated santonin-related compound 2 (SRC2), inhibited NF-κB luciferase reporter activity induced by lymphotoxin ß (LTß) in human lung carcinoma A549 cells. Although SRC2 did not prevent the processing of the NF-κB subunit p100 induced by LTß, it inhibited the nuclear translocation of RelB and p52 in response to the LTß stimulation. In contrast to (-)-dehydroxymethylepoxyquinomicin, SRC2 inhibited the LTß-induced nuclear translocation of the RelB (C144S) mutant in a manner similar to wild-type RelB. While eudesmane derivatives possessing an α-bromoketone moiety or α,ß-unsaturated carbonyl moieties inhibited LTß-induced NF-κB luciferase reporter activity, eudesmane derivatives possessing an α-bromoketone moiety exhibited stronger inhibitory activity on the LTß-induced nuclear translocation of RelB than those possessing a single α-methylene-γ-lactone moiety. The results of the present study revealed that SRC2 inhibits the nuclear translocation of RelB in the non-canonical NF-κB signaling pathway induced by LTß.
Assuntos
Lactonas/farmacologia , Linfotoxina-beta/farmacologia , NF-kappa B/metabolismo , Transporte Proteico/efeitos dos fármacos , Sesquiterpenos de Eudesmano/farmacologia , Células A549 , Núcleo Celular/metabolismo , HumanosRESUMO
BACKGROUND: Exposure to ambient fine particulate matter (PM2.5) is associated with increased cardiometabolic morbidity and mortality. This is widely believed to be attributable to PM2.5 exposure-induced pulmonary and subsequent systemic inflammation. Tumor necrosis factor alpha (TNFα), lymphotoxin α (LTα), and lymphotoxin ß (LTß) are three homologous pro-inflammatory cytokines, each with both unique and redundant activities in inflammation. Their role in PM2.5 exposure-induced inflammation and adverse cardiometabolic effects has to be determined. METHODS AND RESULTS: LTα/TNFα/LTß triple-knockout (TNF/LT KO) and wildtype (WT) mice were exposed to concentrated ambient PM2.5 (CAP) for 5 months. Lung pathological analysis revealed that TNF/LT deficiency reduced CAP exposure-induced pulmonary inflammation. However, glucose homeostasis assessments showed that TNF/LT deficiency significantly aggravated CAP exposure-induced glucose intolerance and insulin resistance. Consistent with glucose homeostasis assessments, CAP exposure significantly increased the body weight and adiposity of TNF/LT KO but not WT mice. In contrast to its body weight effects, CAP exposure reduced food intake of WT but not TNF/LT KO mice. On the other hand, CAP exposure induced marked fat droplet accumulation in brown adipose tissues of WT mice and significantly decreased their uncoupling protein 1 (UCP1) expression, and these effects were markedly exacerbated in TNF/LT KO mice. CONCLUSION: The present study suggests that TNF/LT deficiency influences PM2.5 exposure-induced response of energy metabolism through alterations in both food intake and energy expenditure.