A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.

Leishmania guyanensis suppressed inducible nitric oxide synthase provoked by its viral endosymbiont.

Inducible nitric oxide synthase (iNOS) is essential to the production of nitric oxide (NO), an efficient effector molecule against intracellular human pathogens such as Leishmania protozoan parasites. Some strains of Leishmania are known to bear a viral endosymbiont termed Leishmania RNA virus 1 (LRV1). Recognition of LRV1 by the innate immune sensor Toll-like receptor-3 (TLR3) leads to conditions worsening the disease severity in mice. This process is governed by type I interferon (type I IFNs) arising downstream of TLR3 stimulation and favoring the formation of secondary metastatic lesions. The formation of these lesions is mediated by the inflammatory cytokine IL-17A and occurs in the absence, or low level of, protective cytokine IFN-γ. Here, we described that the presence of LRV1 led to the initial expression of iNOS and low production of NO that failed to control infection. We subsequently showed that LRV1-triggered type I IFN was essential but insufficient to induce robust iNOS induction, which requires strong activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Leishmania guyanensis carrying LRV1 (LgyLRV1+) parasites mitigated strong iNOS production by limiting NF-kB activation via the induction of tumor necrosis factor-alpha-induced protein 3 (TNFAIP3), also known as A20. Moreover, our data suggested that production of LRV1-induced iNOS could be correlated with parasite dissemination and metastasis via elevated secretion of IL-17A in the draining lymph nodes. Our findings support an additional strategy by which LRV1-bearing Leishmania guyanensis evaded killing by nitric oxide and suggest that low levels of LRV1-induced NO might contribute to parasite metastasis.

Níveis Reduzidos de CTRP3 em Pacientes com Doença Arterial Coronariana Estável Relacionados à Presença de Fibrilação Atrial Paroxística / Reduced CTRP3 Levels in Patients with Stable Coronary Artery Disease and Related with the Presence of Paroxysmal Atrial Fibrillation

Resumo Fundamento Os níveis de Proteína 3 relacionada ao fator de necrose tumoral/complemento sérico C1q (CTRP3) e a relação com a fibrilação atrial (FA) na doença arterial coronária estável (DAC) não estão claros atualmente. Objetivos O objetivo deste estudo foi investigar a mudança nos níveis séricos de CTRP3 e sua relação com a FA paroxística em DAC estável. Método O estudo incluiu 252 pacientes com DAC e 50 controles saudáveis com idade/sexo compatíveis. Os níveis séricos de CTRP3 foram medidos, além da anamnese de rotina, exame físico, exames laboratoriais e ecocardiograma. Os pacientes foram divididos em grupos com e sem DAC e indivíduos com DAC com e sem FA paroxística. Os valores eram estatisticamente significativos quando p<0,05. Resultados Os níveis séricos de CTRP3 foram significativamente menores em pacientes com DAC do que no grupo controle (p<0,001). A FA foi detectada em 28 pacientes (15,08%) no grupo DAC. A frequência de hipertensão e do sexo feminino, a proteína C reativa de alta sensibilidade (PCR-as), o nitrogênio ureico no sangue, os níveis de creatinina e o diâmetro diastólico do átrio esquerdo foram maiores (p<0,05 para cada um), e os níveis de CTRP3 foram mais baixos em pacientes com FA (p<0,001). Na análise de regressão logística, os níveis séricos de CTRP3 e os diâmetros diastólicos do átrio esquerdo foram independentemente determinados pelos pacientes com FA (p<0,01 para cada um). Nesta análise, observamos que cada 1 ng/mL de redução nos níveis de CTRP3 aumentou o risco de FA em 10,7%. Na análise ROC dos valores de CTRP3 para detectar pacientes com FA, a área da curva ROC para CTRP3 foi 0,971 (0,951-991) e considerada estatisticamente significativa (p<0,001). Quando o ponto de corte de CTRP3 foi considerado em 300 ng/mL, demonstrava a presença de FA com 87,9% de sensibilidade e 86,8% de especificidade. Conclusão Os níveis séricos de CTRP3 caíram significativamente em pacientes com DAC estável, e níveis reduzidos de CTRP3 estiveram relacionados à presença de FA paroxística nesses pacientes.

Abstract Background Serum Complement C1q/tumor necrosis factor-related protein-3 (CTRP3) levels and the relationship with atrial fibrillation (AF) in stable coronary artery disease (CAD) are not clearly known. Objective The aim of this study was to investigate the change in serum CTRP3 levels and its relationship with paroxysmal AF in stable CAD. Method The study included 252 patients with CAD and 50 age-sex matched healthy control subjects. Serum CTRP3 levels were measured in addition to routine anamnesis, physical examination, laboratory and echocardiography examinations. The patients were divided into groups with and without CAD and CAD patients with and without paroxysmal AF. Statistical significance was accepted as p<0.05. Results Serum CTRP3 levels were found to be significantly lower in patients with CAD than in the control group (p<0.001). AF was detected in 38 patients (15.08%) in the CAD group. The frequency of hypertension and female gender, hs-CRP, blood urea nitrogen, creatinine levels and left atrial end-diastolic (LAd) diameter were higher (p<0.05 for each one), and CTRP3 levels were lower in patients with AF (p <0.001). In the logistic regression analysis, serum CTRP3 levels and LAd diameters were independently determined the patients with AF (p<0.01 for each one). In this analysis, we found that every 1 ng/mL reduction in CTRP3 levels increased the risk of AF by 10.7%. In the ROC analysis of CTRP3 values for detecting patients with AF, the area under the ROC curve for CTRP3 was 0.971 (0.951-991) and was statistically significant (p<0.001). When the CTRP3 cut-off value was taken as 300 ng/mL, it was found to predict the presence of AF with 87.9% sensitivity and 86.8% specificity. Conclusion Serum CTRP3 levels were significantly reduced in patients with stable CAD and decreased CTRP3 levels were closely related to the presence of paroxysmal AF in these patients.

TNFAIP3-upregulated RIP3 exacerbates acute pancreatitis via activating NLRP3 inflammasome.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Accumulating studies have revealed the involvement of tumor necrosis factor alpha-induced protein 3 (TNFAIP3) in the progression of AP. Here, the current study was conducted to elucidate the role of TNFAIP3 and the underlying molecular mechanisms on the progression of AP. The in vivo animal model and in vitro cell model of AP were generated by retrograde injection of sodium taurocholate and stimulation of cerulein into AR42J cells, respectively. Relationships among TNFAIP3, receptor interacting protein 3 (RIP3) and nod-like receptor protein 3 (NLRP3) were predicted on bioinformatics websites and verified by co-immunoprecipitation. AR42J cells were transfected with overexpressing plasmid or shRNA to study the effects of TNFAIP3/RIP3/NLRP3 axis on cell proliferation and apoptosis, secretion of inflammatory cytokines and production of ROS. The effect of TNFAIP3/RIP3/NLRP3 axis in AP was further confirmed in vivo. High expression of TNFAIP3 was observed in AP pancreatic tissues and AP cell model. TNFAIP3 increased RIP phosphorylation through deubiquitination. RIP activated the NLRP3 inflammasome. Silencing of TNFAIP3 or RIP3T led to elevated proliferation and inhibited apoptosis in AR42J cells, accompanied by decreased inflammatory cytokine levels and ROS production. The protective role of inhibited TNFAIP3 in AP was confirmed evidenced by reduced levels of AMY, LIPA, and ROS in vivo. Collectively, overexpressed TNFAIP3 could contribute to the progression of AP by activating RIP3/NLRP3 axis, providing a potential therapeutic target for AP treatment.

Case Report: A Novel TNFAIP3 Mutation Causing Haploinsufficiency of A20 With a Lupus-Like Phenotype.

Genetic mutations that result in loss-of-function of the protein A20 result in an early-onset autoinflammatory disease-haploinsufficiency of A20 (HA20). The reported clinical presentations of HA20 include a Behcet's disease-like phenotype and a more lupus-like phenotype. We have identified a novel mutation in the gene encoding A20 in a pediatric patient with chronic lymphadenopathy, lupus-like symptoms, and progressive hypogammaglobulinemia. This case illustrates the wide range of clinical symptoms, including immunodeficiency, that can occur in patients with HA20.

Activation of the NF-κB Pathway and Heterozygous Deletion of TNFAIP3 (A20) Confer Superior Survival in Extranodal Natural Killer/T-Cell Lymphoma, Nasal Type.

OBJECTIVES: To investigate the role of TNFAIP3 deletions and NF-κB activation in extranodal natural killer/T-cell lymphoma (ENKTCL), nasal type. METHODS: In total, 138 patients with ENKTCL were included. Activation of NF-κB pathway and expression of TNFAIP3 (A20) were examined by immunohistochemistry. TNFAIP3 was analyzed for deletions using FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for investigating neoplasms), for mutations using Sanger sequencing, and for promoter methylation using methylation-specific sequencing. RESULTS: NF-κB pathway activation was observed in 31.2% of cases (43/138), TNFAIP3 expression was negative in 15.2% of cases (21/138), and heterozygous TNFAIP3 deletion was observed in 35% of cases (35/100). TNFAIP3 exons 2 to 9 mutations and promoter methylation were not observed. Kaplan-Meier analysis showed patients with NF-κB pathway activation or TNFAIP3 heterozygous deletion to have a longer overall survival. CONCLUSIONS: Our study demonstrated that NF-κB activation and TNFAIP3 heterozygous deletion confer superior survival in patients with ENKTCL.

The role and mechanism of TNFα-induced protein 3(A20) in phenotypic transformation of human dermal fibroblasts to myofibroblasts / 中国医师杂志

Objective To investigate the role and mechanism of A20 protein in the phenotypic transformation of human dermal fibroblasts (Fb) to myofibroblasts (MFb).Methods Primary human normal skin Fb (NFb) and scar skin Fb (HFb) were isolated and cuhured by enzymatic digestion.The cells were used in logarithmic growth phase at 3-5 passages,and Fb was stimulated by transforming growth factor-β (TGF-β) to mimic the Fb to MFb transformation process.Cell transfection was conducted to knock out the expression of A20 in Fb,and quantitative real-time polymerase chain reaction (PCR) test,protein immunoblotting test were conducted to detect A20,type Ⅰ collagen (Col Ⅰ),type Ⅲ collagen (Col Ⅲ)and α-smooth muscle (α-SMA),and p-Smad 2 expression in Fb.Results Compared with normal skin Fb,A20 was down-regulated in pathological scar Fb (P ＜ 0.05).After TGF-β stimulated,A20 expression was significantly decreased (P ＜ 0.05),while the expression of fibrosis associated molecules such as Col Ⅰ,Col Ⅲ and α-SMA and p-Smad 2 was significantly increased (P ＜ 0.05) in Fb.Knockout of A20 in Fb further promoted TGF-β induced the expression of fibrosis associated molecules such as Col Ⅰ,Col Ⅲ and o-SMA and p-Smad 2 (P ＜ 0.05).Conclusions A20 protein is involved in the process of human dermal Fb to MFb phenotype transformation,and A20 negatively regulates the transformation effect mediated by TGF-β by inhibiting the activation of Smad signaling pathway.

Genetic and Functional Associations with Decreased Anti-inflammatory Tumor Necrosis Factor Alpha Induced Protein 3 in Macrophages from Subjects with Axial Spondyloarthritis.

OBJECTIVE: Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is an anti-inflammatory protein implicated in multiple autoimmune and rheumatologic conditions. We hypothesized that lower levels of TNFAIP3 contributes to excessive cytokine production in response to inflammatory stimuli in axial spondyloarthritis (AxSpA). A further aim was to determine the immune signaling and genetic variation regulating TNFAIP3 expression in individual subjects. METHODS: Blood-derived macrophages from 50 AxSpA subjects and 30 healthy controls were assessed for TNFAIP3 expression. Cell lysates were also analyzed for NF-κB, mitogen-activated protein (MAP) kinase and STAT3 phosphorylation, and supernatants for cytokine production. Coding and regulatory regions in the TNFAIP3 gene and other auto-inflammation-implicated genes were sequenced by next-generation sequencing and variants identified. RESULTS: Mean TNFAIP3 was significantly lower in spondyloarthritis macrophages than controls (p = 0.0085). Spondyloarthritis subject macrophages correspondingly produced more TNF-α in response to lipopolysaccharide (LPS, p = 0.015). Subjects with the highest TNFAIP3 produced significantly less TNF-α in response to LPS (p = 0.0023). Within AxSpA subjects, those on TNF blockers or with shorter duration of disease expressed lower levels of TNFAIP3 (p = 0.0011 and 0.0030, respectively). TNFAIP3 expression correlated positively with phosphorylated IκBα, phosphorylated MAP kinases, and unstimulated phosphorylated STAT3, but negatively with LPS or TNF-α-stimulated fold induction of phosphorylated STAT3. Further, subjects with specific groups of variants within TNFAIP3 displayed differences in TNFAIP3 (p = 0.03-0.004). Nominal pQTL associations with genetic variants outside TNFAIP3 were identified. CONCLUSION: Our results suggest that both immune functional and genetic variations contribute to the regulation of TNFAIP3 levels in individual subjects. Decreased expression of TNFAIP3 in AxSpA macrophages correlated with increased LPS-induced TNF-α, and thus, TNFAIP3 dysregulation may be a contributor to excessive inflammatory responses in spondyloarthritis subjects.

Research progress of the relation between A20 and cancers / 中华内分泌外科杂志

The zinc finger protein A20 exists in many kinds of ceils in the body and plays multiple roles in physiological and pathological processes such as regulation of immune response,inflammatory diseases and cancers by inhibiting cell apoptosis induced by tumor necrosis factor (TNF) and restricting NFKB signaling pathway.Recently,the role of A20 in generation and development of tumors draws wide attention,hence,we summarized recent research progress of the relation between A20 and cancer in this review.