Tumor Necrosis Factor Α-induced Protein 3 Research Articles

Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (β-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

Abstract 52: CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells

Abstract Background: Common failures of chimeric antigen receptor (CAR)-T cell therapy regimens stem from immune escape of target antigen low tumors. Strategies to improve CAR-T cell therapy efficacy include enhancing intrinsic effector function while preventing T cell exhaustion. The ubiquitin-modifying enzyme A20, is a key negative regulator of the NF-kB pathway downstream of T cell receptor activation in T cells. Dysfunctional CD8 T cells in tumors have high expression of A20, and deletion of A20 enhances antitumor activity of CD8 T cells in murine tumor models. We tested whether CRISPR-mediated knock-out (KO) of tumor necrosis factor α-induced protein 3 (TNFAIP3), the gene for A20, increases the effector function and alters the metabolic state of CAR-T cells. Results: A20 deletion led to upregulation of NF-κB target genes and resulted in higher levels of activation marker expression and effector cytokine secretion compared to unarmored CAR-T upon incubation with target cells. In addition, A20 KO CAR-T cells maintained prolonged expression of activation markers upon antigen clearance in the absence of enhanced differentiation, and were more resistant to dysfunction in a repeat challenge serial kill assay. In vivo, A20 deficient CAR-T exhibited increased anti-tumor efficacy against low, medium, and high target expressing tumor models. Consistent with NF-κB upregulation, A20 KO cells significantly upregulated key glycolytic intermediates suggesting that amino acid anabolism was fueled by glycolysis and the pentose phosphate pathway. Moreover, the preferential increase of glycolysis over oxidative phosphorylation confirmed that A20 KO CAR-T cells were skewed towards an effector-like phenotype. Notably, glutathione and its cysteine-glycine bi-products were upregulated upon activation to a higher level in A20 KO CAR-T suggesting enhanced resistance to oxidative stress, in line with their prolonged persistence. Conclusions: CRISPR-mediated knock-out of A20 lowers the activation threshold of CAR-T cells producing enhanced in vitro and in vivo anti-tumor efficacy particularly against tumors with very low levels of target expression. A20 KO induces metabolic rewiring of CAR-T cells by promoting glycolysis and amino acid anabolism to sustain their superior effector functions. This intrinsic armoring strategy can be applied to any CAR-T in any indication to increase tumor control and reduce immune escape. Citation Format: Nina Jia Chu, Carina Nava-Barbero, Ashley Merlino, Ping-Hsien Lee, David Baker, Ben Taylor, Gordon Moody, Letizia Giardino. CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 52.

TNFAIP3 Derived from Skeletal Stem Cells Alleviated Rat Osteoarthritis by Inhibiting the Necroptosis of Subchondral Osteoblasts.

Recent investigations have shown that the necroptosis of tissue cells in joints is important in the development of osteoarthritis (OA). This study aimed to investigate the potential effects of exogenous skeletal stem cells (SSCs) on the necroptosis of subchondral osteoblasts in OA. Human SSCs and subchondral osteoblasts isolated from human tibia plateaus were used for Western blotting, real-time PCR, RNA sequencing, gene editing, and necroptosis detection assays. In addition, the rat anterior cruciate ligament transection OA model was used to evaluate the effects of SSCs on osteoblast necroptosis in vivo. The micro-CT and pathological data showed that intra-articular injections of SSCs significantly improved the microarchitecture of subchondral trabecular bones in OA rats. Additionally, SSCs inhibited the necroptosis of subchondral osteoblasts in OA rats and necroptotic cell models. The results of bulk RNA sequencing of SSCs stimulated or not by tumor necrosis factor α suggested a correlation of SSCs-derived tumor necrosis factor α-induced protein 3 (TNFAIP3) and cell necroptosis. Furthermore, TNFAIP3-derived from SSCs contributed to the inhibition of the subchondral osteoblast necroptosis in vivo and in vitro. Moreover, the intra-articular injections of TNFAIP3-overexpressing SSCs further improved the subchondral trabecular bone remodeling of OA rats. Thus, we report that TNFAIP3 from SSCs contributed to the suppression of the subchondral osteoblast necroptosis, which suggests that necroptotic subchondral osteoblasts in joints may be possible targets to treat OA by stem cell therapy.

METTL14-Mediated m6A Modification of TNFAIP3 Involved in Inflammation in Patients With Active Rheumatoid Arthritis.

The aim of the study was to investigate the role of N6 -methyladenosine (m6A) modification in the progression of rheumatoid arthritis (RA). Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were collected. The expression of m6A modification-related proteins and m6A levels were detected using polymerase chain reaction (PCR), western blot, and m6A enzyme-linked immunosorbent assay (ELISA). The roles of methyltransferase-like 14 (METTL14) in the regulation of inflammation in RA was explored using methylated RNA immunoprecipitation (MeRIP) sequencing and RNA immunoprecipitation assays. Collagen antibody-induced arthritis (CAIA) mice were used as an in vivo model to study the role of METTL14 in the inflammation progression of RA. We found that m6A writer METTL14 and m6A levels were decreased in PBMCs of patients with active RA and correlated negatively with the disease activity score using 28 joint counts (DAS28). Knockdown of METTL14 downregulated m6A and promoted the secretion of inflammatory cytokines interleukin 6 (IL-6) and IL-17 in PBMCs of patients with RA. Consistently, METTL14 knockdown promoted joint inflammation accompanied by upregulation of IL-6 and IL-17 in CAIA mice. MeRIP sequencing and functional studies confirmed that tumor necrosis factor α induced protein 3 (TNFAIP3), a key suppressor of the nuclear factor-κB inflammatory pathway, was involved in m6A-regulated PBMCs. Mechanistic investigations revealed that m6A affected TNFAIP3 expression by regulation of messenger RNA stability and translocation in TNFAIP3 protein coding sequence. Our study highlights the critical roles of m6A on regulation of inflammation in RA progression. Treatment strategies targeting m6A modification may represent a new option for management of RA.

Tumor necrosis factor α-induced protein 3 mediates inflammation and neuronal autophagy in Parkinson’s disease via the NFκB and mTOR pathways

This study aimed to probe the function of tumor necrosis factor α–induced protein 3 (TNFAIP3) in the pathogenesis of Parkinson disease (PD) with its association with autophagy and inflammatory response. TNFAIP3 was reduced in the SN of PD patients (the GSE54282 dataset) and mice and in the MPP+-treated SK-N-SH cells. TNFAIP3 inhibited inflammatory response and enhanced autophagy, thereby alleviating PD in mice. NFκB and mTOR pathways were activated in the SN of PD mice and MPP+-treated cells. TNFAIP3 blocked the two pathways by preventing the p65 nuclear translocation and stabilizing DEPTOR, an endogenous inhibitor of mTOR. NFκB activator LPS and mTOR activator MHY1485 reversed the effects of TNFAIP3 on mitigation of injury in PD mice and in SK-N-SH cells induced with MPP+. Altogether, TNFAIP3 played a neuroprotective role in MPTP-induced mice by restricting NFκB and mTOR pathways.

A20 haploinsufficiency caused by loss‐of‐function TNFAIP3 mutation likely leads to progression of antiphospholipid syndrome to marginal zone lymphomas following coronavirus disease 2019 vaccination: A case study

AbstractAntiphospholipid syndrome (APS) is a rare autoimmune systemic disorder. Previously, no report suggests that APS could progress to extranodal marginal zone lymphomas (EMZL). In this study, we met an unusual APS patient with such progression to EMZL. The patients had been diagnosed with APS two years ago and was in a stable condition after regular treatment until his readmission to our hospital and re‐diagnosed with EMZL recently. Coincidentally, we noticed that the patient had been immunized against inactivated COVID‐19 vaccine just 2 days before his readmission. Furthermore, we performed whole‐exome sequencing and identified a heterozygous, new variant in TNFAIP3 (tumor necrosis factor, α‐induced protein 3) which encoded A20 protein, a key molecule controlling NF‐κB signaling. This variation caused a loss of a base A in TNFAIP3 gene at position 443_444, leading to a frameshift mutation and the production of a truncated A20 Lys148fs*67. A20 Lys148fs*67 failed to suppress TNF‐α‐induced NF‐κB activation and might act through haploinsufficiency. Vaccines work by triggering an immune response to a virus or bacterium within the body. A20 negatively regulates NF‐κB signaling to protect immune system from overactivation. In our case, the newly identified mutation in the TNFAIP3 led to the production of a loss‐of‐function A20 Lys148fs*67, which lost the ability to inhibit inflammation. The patient with such a heterozygous mutation, when facing with the “second hit” of COVID‐19 vaccination challenge, might produce excessive amounts of inflammatory cytokines and formed "cytokine storm" duo to A20 haploinsufficiency, eventually leading to the progression from APS to EMZL.

The "time-response" effect of Wenyang Pingchuan Formula on miR-19a in asthmatic mice experiment.

Wenyang Pingchuan Formula (WPCF) is an empirical formula for the treatment of acute childhood asthma. However, the "time-effect" relationship of this prescription is not clear. This paper explores the relationship between Janus activated kinase signal transducer and activator of transcriptions (JAK/STAT), nuclear factor-κB (NF-κB), and microRNA (miR-19a), and also preliminarily determines the best time-effect relationship of WPCF in reducing the airway inflammation in asthmatic mice. 80 BALB/c mice were randomly divided into four groups: control (CON) group, model (MDL) group, dexamethasone (DEX) group, and WPCF group. MDL group was established through intraperitoneal injection of 10% ovalbumin (OVA) and Al(OH)3 solution and the inhalation of aerosolized 5% OVA solution. Enzyme-linked immunosorbent assay (ELISA), real-time PCR and Western blot were conducted to determine the levels of interleukin (IL)-4, IL-13, interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF), contents of miR-19a mRNA and STAT6, phosphorylated signal transducers and activators of transcription 6 (p-STAT6), p65, phosphorylated p65 (p-p65), suppressors of cytokine signaling 1 (SOCS1), and tumor necrosis factor α-induced protein-3 (Tnfaip3) proteins after 7 and 28 days of intervention respectively. Significant down-regulation of IL-4 and IL-13 expression (P<0.05) and up-regulation of IFN-γ expression (P<0.05) in BALF have been observed for WPCF group compared with the MDL group. The significant down-regulation of miR-19a mRNA and STAT6, p-STAT6, p65, p-p65 proteins (P<0.05) and up-regulation of SOCS1 and Tnfaip3 proteins (P<0.05) in BALF was also observed for WPCF group compared to the MDL group. During the experiment, the weight of the mice in DEX group significantly decreased (P<0.05) compared with the other groups. WPCF could restore Th1/Th2 balance. The longer the intervention time, the more effective the treatment. The down-regulation of miR-19a mRNA by activating JAK/STAT and NF-κB signal pathways may be a possible mechanism by which WPCF alleviates airway inflammation.

TNFAIP3 ameliorates the degeneration of inflammatory human nucleus pulposus cells by inhibiting mTOR signaling and promoting autophagy.

Autophagy is involved in degenerative diseases such as osteoarthritis and disc degeneration. Although, tumor necrosis factor α-induced protein 3 (TNFAIP3) is well-known as a key regulator of inflammation and autophagy, it is still not clear whether TNFAIP3 regulates autophagy to protect from human disc cells degeneration. We hypothesize that TNFAIP3 may also regulate autophagy to inhibit pro-inflammatory cytokines expression in human nucleus pulposus cells (NPCs). In this study, TNFAIP3 expression was increased in degenerative disc tissue as well as LPS-stimulated human NPCs, and the effect of TNFAIP3 in LPS-induced NPCs was further explored. The results demonstrated that pro-inflammatory cytokines expression in TNFAIP3-His cells was decreased, while it was increased in TNFAIP3-siRNA cells. Further molecular mechanism research showed that TNFAIP3-siRNA cells enhanced the phosphorylation of mammalian target of rapamycin (mTOR) and inhibited autophagy. Meanwhile, after treatment of TNFAIP3-siRNA cells with the mTOR inhibitor Torin1, the level of autophagy increased and the decrease of extracellular matrix was reversed. In summary, overexpressed TNFAIP3 can promote autophagy and reduce inflammation in LPS-induced human NPCs. Moreover, autophagy triggered by TNFAIP3 can ameliorate the degeneration of inflammatory human NPCs, providing a potential and an attractive therapeutic strategy for degenerative disease.

Sikyungbanha-Tang Suppressing Acute Lung Injury in Mice Is Related to the Activation of Nrf2 and TNFAIP3.

Sikyungbanha-Tang (SKBHT) is a Chinese traditional medicine popularly prescribed to patients with respiratory inflammatory symptoms in Korea. Although the Korea Food and Drug Administration approved SKBHT as a therapeutics for relieving the symptoms, experimental evidence for SKBHT suppressing inflammation is scarce. Here, we presented evidence that SKBHT can suppress inflammation in an acute lung injury (ALI) mouse model and explored the possible underlying mechanisms of SKBHT's anti-inflammatory activity. Single intratracheal (i.t.) injection of SKBHT (1 mg/kg or 10 mg/kg body weight) into mouse lungs decreased prototypic features of lung inflammation found in ALI, such as a high level of proinflammatory cytokines, neutrophil infiltration, and the formation of hyaline membrane, which were induced by a single i.t. LPS (2 mg/kg body weight). When added to a murine macrophage RAW 264.7 cells, SKBHT activated an anti-inflammatory factor Nrf2, increasing the expression of genes regulated by Nrf2. SKBHT suppressed the ubiquitination of Nrf2, suggesting that SKBHT increases the level of and thus activates Nrf2 by blunting the ubiquitin-dependent degradation of Nrf2. SKBHT induced the expression of tumor necrosis factor α-induced protein 3 (TNFAIP3), an ubiquitin-modulating protein that suppresses various cellular signals to NF-κB. Concordantly, SKBHT suppressed NF-κB activity and the expression of inflammatory cytokine genes regulated by NF-κB. Given that Nrf2 and TNFAIP3 are involved in regulating inflammation, our results suggest that SKBHT suppresses inflammation in the lung, the effect of which is related to SKBHT activating Nrf2 and TNFAIP3.

MiR‐605‐5p promotes invasion and proliferation by targeting TNFAIP3 in non–small‐cell lung cancer

Lung cancer is an significant cause of death worldwide, and non-small-cell lung cancer (NSCLC) is the most common type of lung cancer. MicroRNAs (miRNAs) have been identified to play key roles in NSCLC development. Recently, it has beenreported that miR-605-5p is a cancer-related miRNA in several types of tumors. In this study, we study the role of miR-605-5p in NSCLC cells. We find that miR-605-5p is upregulated in NSCLC cells. Overexpression of miR-605-5p significantly promotes lung cancer invasion and migration in H460 and H1299 cells. Besides this, miR-605-5p also promotes lung cancer cell carcinoma proliferation and metastasis in vivo. However, downregulation of miR-605-5p inhibitscell invasion and migration byinhibiting lung cancer cell carcinoma proliferation and metastasis. In addition,the luciferase report assay identifies 3'-untranslated region tumor necrosis factor α-induced protein 3 (TNFAIP3) asa target of miR-605-5p. Silencing of TNFAIP3 promotesinvasion and proliferation in lung cancer. In addition, the knockdown of TNFAIP3 restores the significant decrease in invasion and proliferation in miR-605-5p-inhibitor-transfected lung cancer cells. In conclusion, miR-605-5p promotes invasion and proliferation by targeting TNFAIP3 in NSCLC, andmay provide possible biomarkers for NSCLC therapy.

TNFAIP3 genetic polymorphisms reduce ankylosing spondylitis risk in Eastern Chinese Han population

This study was conducted to clarify the associations of tumor necrosis factor-α induced protein 3 (TNFAIP3) and TNFAIP3-interacting protein 1 (TNIP1) genetic polymorphisms with ankylosing spondylitis (AS) susceptibility. Five single nucleotide polymorphisms (SNPs) in TNFAIP3 gene and four in TNIP1 gene were genotyped in 667 AS patients and 667 matched healthy controls. Genotypes and haplotype analysis were conducted by using SPSS 23.0 and Haploview 4.2 software. The T allele and CT genotype in TNFAIP3 rs10499194 were significantly associated with a reduced AS risk (T allele vs. C allele, OR = 0.619, 95% CI = 0.430–0.889, P = 0.009; CT vs. CC, OR = 0.603, 95% CI = 0.416–0.875, P = 0.007). However, no association remained significant after Bonferroni correction. The rs13207033A- rs10499194T haplotype of TNFAIP3 conferred a protective effect on AS susceptibility. Stratification analyses suggested that rs10499194 polymorphism decreased the risk of AS in the male subgroup, subgroup aged ≥ 29, HLA-B27 positive subgroup as well as the subgroups of BASFI < 4 and BASDAI < 4 (all P < 0.05). Furthermore, the functional annotation suggested a potential function of rs10499194 mutation. Our results demonstrated that TNFAIP3 rs10499194 polymorphism may be associated with a reduced risk of AS.

A20 Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells to 60Co-γ Ionizing Radiation

The radioresistance of hepatocellular carcinoma (HCC) cells is a critical obstacle for effectively applying radiotherapy (RT) in HCC treatment. NF-κB, an important transcription factor, can influence critical cell fate decisions by promoting cell survival or anti-apoptosis in response to cell-stress, e.g. chemotherapies or ionizing radiation (IR). A20, also named as tumor necrosis factor α induced protein 3 (TNFAIP3 ), is a dominant negative regulator of NF-κB pathway and its functions in HCC are largely unknown. Cell culture and adenovirus vectors L-02, HepG2, MHCC-97H (a highly aggressive HCC cell line) and MHCC-97L (a lowly aggressive HCC cell line) cells were purchased from cell resources center of Chinese Academy of Medical Sciences (Beijing, China). The adenovirus vectors containing full length sequence of A20 and its A20 siRNA was purchased from Vigene Company (Jinan, Shandong Province, China). The HCC specimens and the adjacent non-tumor tissues were obtained by routinely surgical operations and preserved by our lab. The collection of tissues and the study protocol was all approved by the Ethics Committee of Xi’an Jiaotong University School of Medicine, and with the informed consent of patients. Paired HCC and non-tumorous tissues (40 paired) were obtained from surgical resections from February 2015 to April 2016. Using such methods as qPCR; Western Blot analysis; 60Co-γ ionizing radiation and colony formation; TUNEL reaction; Apoptosis analysis; Transwell analysis; In vivo tumor growth. Higher expression of A20 was detected in hepatic non-tumor cell line or clinical specimens compared with HCC cell lines or clinical specimens. A20 decreased the expression of proteins mediating cellular stress/injury response or epithelial-mesenchymal transition (EMT) process. Overexpression of A20 via adenovirus enhanced the effect of 60Co-γ ionizing radiation (IR) on HCC cells’ injury, e.g. G2/M arrest or DNA double strands break (DSB). Moreover, A20 also enhanced the in vitro or in vivo survival inhibiting of HCC cells induced by IR. Our results showed A20 downregulates the protein level of cellular stress/injury response regulators and by then potentiate the susceptibility of HCC cell line to 60Co-γ IR. Overexpression of A20 via an adenoviral vector can be a useful strategy for increasing tumor radiosensitivity.

Decreased MiR-128-3p alleviates the progression of rheumatoid arthritis by up-regulating the expression of TNFAIP3.

Background: Rheumatoid arthritis (RA) is a inflammatory disease that characterized with the destruction of synovial joint, which could induce disability. Inflammatory response mediated the RA. It has been reported that MiR-128-3p is significantly increased in RA, while the potential role was still unclear.Methods: T cells in peripheral blood mononuclear cell (PBMC) were isolated from the peripheral blood from people of RA and normal person were used. Real-time PCR was performed to detect the expression of MiR-128-3p, while the protein expression of tumor necrosis factor-α-induced protein 3 (TNFAIP3) was determined using Western blot. The levels of IL-6 and IL-17 were measured using enzyme-linked immunosorbent assay (ELISA). The expression of CD69 and CD25 was detected using flow cytometry. The RA mouse model was constructed for verification of the role of MiR-128-3p.Results: The expression of MiR-128-3p was significantly increased, while TNFAIP3 was decreased, the levels of IL-6 and IL-17 were also increased in the T cells of RA patients. Down-regulated MiR-128-3p significantly suppressed the expression of p-IkBα and CD69, and CD25in T cells. MiR-128-3p targets TNFAIP3 to regulate its expression. MiR-128-3p knockdown significantly suppressed the activity of nuclear factor κB (NF-κB) and T cells by up-regulating TNFAIP3, while cells co-transfected with si-TNFAIP3 abolished the effects of MiR-128-3p knockdown. The in vivo experiments verified the potential role of MiR-128-3p on RA.Conclusion: Down-regulated MiR-128-3p significantly suppressed the inflammation response of RA through suppressing the activity of NF-κB pathway, which was mediated by TNFAIP3.

Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis.

De novo autoimmune hepatitis (DAIH) is an important cause of late allograft dysfunction following liver transplantation, but its cause and underlying pathogenesis remains unclear. We sought to identify specific innate and adaptive immune mechanisms driving the pro-inflammatory cytokine secreting regulatory T cell (Treg) phenotype in DAIH and determine if modulation of these pathways could resolve the inflammatory milieu observed in the livers of patients with DAIH. Here, we demonstrate toll-like receptors (TLRs) 2- and 4-mediated inflammasome activation in CD14++ monocytes, a finding that is key to maintaining dysfunctional Tregs in patients with DAIH. Furthermore, silencing of TLR 2 and 4 in CD14++ monocytes prevented activation of the inflammasome and significantly decreased IFN-γ production by FOXP3+ Tregs. We also observed significantly increase in expression of tumor necrosis factor α-induced protein 3 (TNFAIP3), a negative regulator of the NLRP3 Inflammasome, in monocytes/macrophages of liver transplant subjects who have normal allograft function and do not have DAIH. TNFAIP3 expression was virtually absent in monocytes/macrophages of patients with DAIH. Our findings suggest that autoimmunity in DAIH is promoted by CD14++ monocytes predominantly through activation of inflammatory signaling pathways.

A20 enhances the radiosensitivity of hepatocellular carcinoma cells to 60Co-γ ionizing radiation.

The radioresistance of hepatocellular carcinoma (HCC) cells is a critical obstacle for effectively applying radiotherapy (RT) in HCC treatment. NF-κB, an important transcription factor, can influence critical cell fate decisions by promoting cell survival or anti-apoptosis in response to cell-stress, e.g. chemotherapies or ionizing radiation (IR). A20, also named as tumor necrosis factor α induced protein 3 (TNFAIP3), is a dominant negative regulator of NF-κB pathway and its functions in HCC are largely unknown. The present work aimed to reveal the role of A20 plays in affecting the radiosensitivity of HCC cells. Higher expression of A20 was detected in hepatic non-tumor cell line or clinical specimens compared with HCC cell lines or clinical specimens. A20 decreased the expression of proteins mediating cellular stress/injury response or epithelial-mesenchymal transition (EMT) process. Overexpression of A20 via adenovirus enhanced the effect of 60Co-γ ionizing radiation (IR) on HCC cells’ injury, e.g. G2/M arrest or DNA double strands break (DSB). Moreover, A20 also enhanced the in vitro or in vivo survival inhibiting of HCC cells induced by IR. These results reveal the roles of A20 in HCC radiosensitization and overexpression of A20 would be a novel strategy for HCC radiotherapy.

TNFAIP3 gene rs7749323 polymorphism is associated with late onset myasthenia gravis.

In this study, we intended to genotype 2 single nucleotide polymorphisms (SNPs) of tumor necrosis factor α-induced protein 3 (TNFAIP3) genes and explore an association of TNFAIP3 genetic polymorphism with the patients of myasthenia gravis (MG) at clinical level. In brief, 215 of adult MG patients were divided into subgroups according to their clinical features, age of onset, thymic pathology, and autoantibodies. Two hundred thirty-five of healthy controls were also divided into subgroups with gender- and age-matched. The allele and genotype frequencies of subgrouped patients were found to be higher than those of healthy controls. The distribution of TNFAIP3 gene rs7749323∗A allele of late onset MG (LOMG, with positive acetylcholine receptor antibody and without thymoma) subgrouped patients was also significantly higher than that of gender- and age-matched healthy controls (7.4% vs 2.4%, odds ratio [OR] = 3.27, 95% confidence interval [CI] 1.01–10.6, P = .04). Furthermore, analysis to the genotype frequencies indicates that the carriers of rs7749323∗A allele of LOMG group became more frequent than that of age-matched healthy controls (14.9% vs 4.8%, OR = 3.47, 95% CI 1.04–11.6, dominant model: P = .03). It is interesting to notice that there is no significant association between the rs7749323 and susceptibility of other MG subgroups. Therefore, it is suggested that the SNPs in the 3′ flanking region (rs7749323) of TNFAIP3 gene and the genetic variations of TNFAIP3 gene may take an important role in the susceptibility of LOMG.

TNFAIP3 overexpression is an independent factor for poor survival in esophageal squamous cell carcinoma.

Tumor necrosis factor α induced protein3 (TNFAIP3) is a protein that is induced by TNF-mediated NF-κB activation and has a dual function in regulating NF-κB. TNFAIP3 is associated with inflammatory carcinogenesis in many cancer types. However, the clinical significance of TNFAIP3 expression and function in esophageal squamous cell carcinoma (ESCC) has not yet been reported. We examined 149ESCC tissue specimens to determine the clinical significance of TNFAIP3 by immunohistochemistry. Western blot analyses were used to detect TNFAIP3 expression in TE-1, TE-8, TE-15 and KYSE-70 ESCC cells and in Het-1A, a non-cancerous esophageal cell line. TNFAIP3 protein knockdown was conducted using small-interfering RNA to investigate its impact on cell proliferation, migration and invasion. Significant correlations between TNFAIP3 expression and differentiation (P=0.04) among clinicopathological characteristics of ESCC patients were demonstrated, and high TNFAIP3 expression was associated with poor survival (P=0.02). Moreover, multivariate analysis result showed that high TNFAIP3 expression was an independent factor for poor survival (P=0.04). In vitro analysis showed high expression of TNFAIP3 protein in TE-15 cells and low expression in Het-1A cells. Furthermore, the proliferation, migration and invasion of TE-15 cells after TNFAIP3 suppression by siRNA were significantly reduced. These findings suggest that TNFAIP3 protein may be an independent prognostic marker for poor survival, and a promising target for ESCC therapy.

C/EBP β mRNA expression is upregulated and positively correlated with the expression of TNIP1/TNFAIP3 in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

CCAAT/enhancer-binding protein β (C/EBP β) has important roles in numerous signaling pathways. The expression of the majority of regulators and target gene products of C/EBP β, including tumor necrosis factor α-induced protein 3 (TNFAIP3) and TNFAIP3-interacting protein 1 (TNIP1), are upregulated in patients with systemic lupus erythematosus (SLE). The aim of the present study was to investigate whether C/EBP β expression is associated with SLE pathogenesis and correlates with TNIP1 and TNFAIP3 expression. Quantitative reverse transcription-polymerase chain reaction analysis was used to assess the expression of C/EBP β, TNIP1, and TNFAIP3 mRNA in peripheral blood mononuclear cells (PBMC) from 20 patients with SLE and 20 healthy controls. Spearman's rank test was used to determine the correlation between C/EBP β expression and SLE disease activity, and that between C/EBP β expression and TNIP1/TNFAIP3 expression in PBMCs from patients with SLE. C/EBP β mRNA expression was markedly increased in patients with SLE compared with healthy controls. The expression of C/EBP β was positively correlated with the SLE disease activity index and negatively correlated with the serum level of complement components C3 and C4. In addition, C/EBP β mRNA expression was increased in PBMCs from SLE patients that were positive for antinuclear, anti-Smith and anti-nRNP antibodies, compared with the antibody negative SLE patients. Furthermore, the mRNA expression levels of C/EBP β in patients with SLE was positively correlated with TNIP1 and TNFAIP3 expression. The results of the current study suggest that the increased expression of C/EBP β in PBMCs and the interaction between C/EBP β and TNIP1/TNFAIP3 may be involved in the pathogenesis of SLE.

Tumor necrosis factor α-induced protein-3 protects zinc transporter 8 against proinflammatory cytokine-induced downregulation.

Zinc transporter 8 (ZnT8) is exclusively expressed in the pancreatic islet and is essential for insulin crystallization, hexamerization and secretion. Tumor necrosis factor α-induced protein-3 (TNFAIP3) is a zinc finger protein that serves a major role in the negative feedback regulation of NF-κB signaling in response to multiple stimuli, and is a central regulator of immunopathology. Although the role of TNFAIP3 in diabetes has been extensively studied, its effect on ZnT8 has not been fully elucidated. The present study aimed to verify whether proinflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), are able to affect ZnT8 expression in islet cells. In addition, the study aimed to determine the effect of TNFAIP3 overexpression on cytokine-altered ZnT8 activity, considering its effect on NF-κB signaling. Cell-based studies using NIT-1 cells overexpressing TNFAIP3 were used to assess the effect of cytokines on ZnT8 and NF-κB activation, as well as the effect of TNFAIP3 on ZnT8 expression. Western blot analysis and immunofluorescence staining were employed to determine the protein expression and NF-κB activation, respectively. The results indicated that cytokine stimulation led to TNFAIP3 upregulation, ZnT8 downregulation and NF-κB activation. Furthermore, TNFAIP3 overexpression protected ZnT8 from cytokine-induced downregulation. In conclusion, the current results suggest that inflammation or TNFAIP3 dysfunction may be involved in the pathogenesis of diabetes via ZnT8 expression, besides from islet cell apoptosis. In addition, restricting inflammation and enhancing TNFAIP3 expression may exert a positive effect in diabetes prevention, treatment and pancreatic cell transplantation.

Association analysis between TNIP1 gene polymorphisms and systemic lupus erythematosus in a Chinese Han population

Objective To investigate association between polymorphisms of the tumor necrosis factor α-induced protein 3 (TNFAIP3) interacting protein 1 (TNIP1) gene and systemic lupus erythematosus (SLE) in a Chinese Han population. Methods Blood samples were collected from 284 patients with SLE of Han nationality (SLE group) and 630 healthy controls of Han nationality (control group). Ligase detection reaction (LDR) was performed to determine the genotypes of 120 single nucleotide polymorphisms (SNPs) in the TNIP1 gene. Data were analyzed with the PLINK 1.07 package and Haploview software. Results After quality control, data on 105 SNPs underwent statistical analysis finally. Four SNPs including rs3805433, rs12516176, rs6869605 and rs4958882 in the TNIP1 gene were significantly associated with SLE (OR: 1.50-1.53, all P<4.72 × 104) , and there was a significant increase in the frequency of rs3805433 C, rs12516176 C, rs6869605 C and rs4958882 G alleles in the SLE group (0.301-0.306) compared with the control group (0.221-0.225). There was strong linkage disequilibrium between these 4 SNPs (r2 ≥ 0.871, D′ ≥ 0.938). In addition, moderate linkage disequilibrium was observed between these 4 SNPs and a previously reported SLE-related SNP rs10036748 (r2 ≥ 0.073, D′ ≥ 0.868). The frequency of the haplotype H2: CCCGT was significantly higher in the SLE group than in the control group (0.290 vs. 0.210, OR=1.54, P<4.72 × 10-4). Conclusion TNIP1 gene polymorphisms are associated with SLE in Chinese Han population. Key words: Lupus erythematosus, systemic; Polymorphism, single nucleotide; Genetic association studies; Genes, TNIP1