A20 (TNFAIP3) alleviates viral myocarditis through ADAR1/miR-1a-3p-dependent regulation.

OBJECTIVE: To investigate the effect of A20 and how A20 is regulated in viral myocarditis (VMC). METHODS: BABL/C mice, primary neonatal rat cardiomyocytes and H9c2 cells were infected with Coxsackie virus B3 (CVB3) to establish animal and cellular models of VMC. H&E staining revealed the pathologic condition of myocardium. ELISA measured the serum levels of creatine kinase, creatine kinase isoenzyme and cardiac troponin I. The effects of A20, miR-1a-3p and ADAR1 were investigated using gain and loss of function approaches. ELISA measured the levels of IL-6, IL-18 and TNF-α in serum or cell culture supernatant. TUNEL staining and flow cytometry assessed the apoptosis of myocardium and cardiomyocytes, respectively. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays verified the binding between A20 and miR-1a-3p. Co-immunoprecipitation assay verified the binding between ADAR1 and Dicer. RESULTS: A20 was underexpressed and miR-1a-3p was overexpressed in the myocardium of VMC mice as well as in CVB3-infected cardiomyocytes. Overexpression of A20 suppressed cardiomyocyte inflammation and apoptosis in vivo and in vitro. miR-1a-3p promoted CVB3-induced inflammation and apoptosis in cardiomyocytes by binding to A20. The expression of miR-1a-3p was regulated by ADAR1. ADAR1 promoted the slicing of miR-1a-3p precursor by binding to Dicer. CONCLUSION: A20, regulated by ADAR1/miR-1a-3p, suppresses inflammation and cardiomyocyte apoptosis in VMC.

A20 regulates the therapeutic effect of anti-PD-1 immunotherapy in melanoma.

BACKGROUND: The therapeutic effect of immune checkpoint blockers, especially the neutralizing antibodies of programmed cell death (PD-1) and its ligand programmed death ligand 1 (PD-L1), has been well verified in melanoma. Nevertheless, the dissatisfactory response rate and the occurrence of resistance significantly hinder the treatment effect. Inflammation-related molecules like A20 are greatly implicated in cancer immune response, but the role of tumorous A20 in antitumor immunity and immunotherapy efficacy remains elusive. METHODS: The association between tumorous A20 expression and the effect of anti-PD-1 immunotherapy was determined by immunoblotting, immunofluorescence staining and flow cytometry analysis of primary tumor specimens from melanoma patients. Preclinical mouse model, in vitro coculture system, immunohistochemical staining and flow cytometry analysis were employed to investigate the role of A20 in regulating the effect of anti-PD-1 immunotherapy. Bioinformatics, mass spectrum analysis and a set of biochemical analyzes were used to figure out the underlying mechanism. RESULTS: We first discovered that upregulated A20 was associated with impaired antitumor capacity of CD8+T cells and poor response to anti-PD-1 immunotherapy in melanoma patients. Subsequent functional studies in preclinical mouse model and in vitro coculture system proved that targeting tumorous A20 prominently improved the effect of immunotherapy through the invigoration of infiltrating CD8+T cells via the regulation of PD-L1. Mechanistically, A20 facilitated the ubiquitination and degradation of prohibitin to potentiate STAT3 activation and PD-L1 expression. Moreover, tumorous A20 expression was highly associated with the ratio of Ki-67 percentage in circulating PD-1+CD8+T cells to tumor burden. CONCLUSIONS: Together, our findings uncover a novel crosstalk between inflammatory molecules and antitumor immunity in melanoma, and highlight that A20 can be exploited as a promising target to bring clinical benefit to melanomas refractory to immune checkpoint blockade.

Keratinocyte Expression of A20/TNFAIP3 Controls Skin Inflammation Associated with Atopic Dermatitis and Psoriasis.

Keratinocytes are key players in chronic inflammatory skin diseases. A20 regulates NF-κB-dependent expression of proinflammatory genes and cell death, but the impact of its expression in keratinocytes on systemic inflammation and skin disorders has not been determined. Comparative transcriptomic analysis of microdissected epidermis showed that A20 is down-regulated in involved epidermis, but not in dermis, of psoriasis and atopic dermatitis patients, suggesting that loss of A20 expression in keratinocytes increases the vulnerability for psoriasis/atopic dermatitis induction. We have previously shown that epidermis-specific A20 knockout mice (A20EKO) develop mild epidermal hyperplasia but no macroscopic skin inflammation. We now show that various cytokines and chemokines are up-regulated in A20EKO mouse skin. A20EKO mice also display systemic proinflammatory changes, even in the absence of skin immune cell infiltration, and an exacerbated disease severity upon induction of experimental psoriasis, atopic dermatitis, or skin barrier disruption. Keratinocytes showed increased proinflammatory gene expression in the absence of A20 in unstimulated and IL-17A-stimulated conditions, in part resulting from uncontrolled MyD88-dependent signaling. Our findings indicate that absence of A20 in keratinocytes leads to systemic inflammation at homeostatic conditions and is sufficient to exacerbate inflammatory skin disorders associated with different immune profiles by increasing cytokine and chemokine expression.

The pleiotropic effects of TNFα in breast cancer subtypes is regulated by TNFAIP3/A20.

TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.

M2b macrophages reduce early reperfusion injury after myocardial ischemia in mice: A predominant role of inhibiting apoptosis via A20.

BACKGROUND: Monocytes or macrophages have been assessed as potential therapeutics to ameliorate myocardial ischemic diseases, but the results have been controversial. As regulatory macrophages, M2b macrophages could have enhanced protective effects. We tested the hypothesis that transplantation of M2b macrophages could ameliorate myocardial ischemia/reperfusion (I/R) injury. The potential mechanisms involved in it were investigated. METHODS: M2b macrophages were polarized by lipopolysaccharide (LPS) and the immune complex (IC) from bone marrow-derived macrophages (BMDMs) of C57BL/6 mice. They were identified based on surface marker expression and cytokine production. Myocardial I/R injury models were established with the same strain of mice. Once the ischemic area was identified, either 1×105 M2b macrophages (MT group) or the same volume of normal saline (CK group) was injected into the ischemic zone. Mice in the sham operation (SO) group underwent the operation without ligation of the coronary artery. RESULTS: We found a significant decrease in serum cardiac troponin I (cTnI) level, the infarct area, apoptosis index, and nuclear factor-κB (NF-κB) signaling activation in the MT group after 2h of reperfusion; the changes were induced by I/R. In addition, the injury resulted in significantly up-regulated expression of A20 and continued to be improved by the transplanted M2b macrophages. CONCLUSIONS: The administration of M2b macrophages significantly attenuated myocardial I/R injury. A20 may be part of the protective mechanism through limiting NF-κB signaling-mediated apoptosis.

Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

In visceral leishmaniasis, we found that the antileishmanial drug Amp B produces a higher level of IL-1ß over the infected control. Moreover, administering anti-IL-1ß antibody to infected Amp B-treated mice showed significantly less parasite clearance. Investigation revealed that Leishmania inhibits stimuli-induced expression of a multiprotein signaling platform, NLRP3 inflammasome, which in turn inhibits caspase-1 activation mediated maturation of IL-1ß from its pro form. Attenuation of NLRP3 and pro-IL-1ß in infection was found to result from decreased NF-κB activity. Transfecting infected cells with constitutively active NF-κB plasmid increased NLRP3 and pro-IL-1ß expression but did not increase mature IL-1ß, suggesting that IL-1ß maturation requires a second signal, which was found to be reactive oxygen species (ROS). Decreased NF-κB was attributed to increased expression of A20, a negative regulator of NF-κB signaling. Silencing A20 in infected cells restored NLRP3 and pro-IL-1ß expression, but also increased matured IL-1ß, implying an NF-κB-independent A20-modulated IL-1ß maturation. Macrophage ROS is primarily regulated by mitochondrial uncoupling protein 2 (UCP2), and UCP2-silenced infected cells showed an increased IL-1ß level. Short hairpin RNA-mediated knockdown of A20 and UCP2 in infected mice independently documented decreased liver and spleen parasite burden and increased IL-1ß production. These results suggest that Leishmania exploits A20 and UCP2 to impair inflammasome activation for disease propagation.-Gupta, A. K., Ghosh, K., Palit, S., Barua, J., Das, P. K., Ukil, A. Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

IL-17RC is critically required to maintain baseline A20 production to repress JNK isoform-dependent tumor-specific proliferation.

The IL-17/IL-17R axis has controversial roles in cancer, which may be explained by tumor-specific results. Here, we describe a novel molecular mechanism underlying IL-17RC-controlled tumor-specific proliferation. Triggered by IL-17RC knockdown (KD), B16 melanoma and 4T1 carcinoma cells inversely altered homeostatic tumor proliferation and tumor growth in vitro and in vivo. In contrast to the existing dogma that IL-17RC-dependent signaling activates the JNK pathway, IL-17RC KD in both tumor cell lines caused aberrant expression and activation of different JNK isoforms along with markedly diminished levels of the ubiquitin-editing enzyme A20. We demonstrated that differential up-regulation of JNK1 and JNK2 in the two tumor cell lines was responsible for the reciprocal regulation of c-Jun activity and tumor-specific proliferation. Furthermore, we showed that A20 reconstitution of IL-17RCKD clones with expression of full-length A20, but not a truncation-mutant, reversed aberrant JNK1/JNK2 activities and tumor-specific proliferation. Collectively, our study reveals a critical role of IL-17RC in maintaining baseline A20 production and a novel role of the IL-17RC-A20 axis in controlling JNK isoform-dependent tumor-specific homeostatic proliferation.

The Effects of miR-136-5p-Mediated Regulation of A20 in Astrocytes from Cultured Spinal Cord Cultured Cells In Vitro.

BACKGROUND/AIMS: This study focused on investigating the regulatory mechanism of miR-136-5p in mouse astrocytes stimulated with interleukin-17(IL-17). METHODS: C57BL/6 mouse astrocytes were stimulated with IL-17 (100ng/ml) for various periods of time (0-48 hours) and at various doses (0-200 ng), and the expression levels of inflammatory cytokine and chemokine genes (IL-6, TNF-α, MCP-1, MCP-5 and MIP-2) were then detected by real-time PCR. The expression of the A20 gene was measured with real-time PCR in cells that were stimulated with IL-17 (50 ng/ml) for various periods of time (0-48 hours). C57BL/6 mouse astrocytes were transfected with Ctrl-anti-miR-136-5p or LNA -anti-miR-136-5p for 48 h. Thereafter, the cells were stimulated with or without IL-17 (50ng/ml) for 6 h. The level of A20 protein (TNFα-induced protein 3, TNFAIP3) was detected by Western blot analysis. RESULTS: (1) Compared with the DMEM control group, within six hours, IL-17 stimulation significantly increased the expression levels of inflammatory cytokine and chemokine genes and clearly decreased the expression level of the A20 protein. (2) Without IL-17 stimulation, the expression level of the miR-136-5p gene was significantly decreased, whereas in the miR-136-5p-inhibition group, the A20 protein expression was elevated. IL-17 stimulation slightly decreased the expression of the A20 protein in the miR-136-5p-inhibition group, but it was still slightly higher than in the control group. CONCLUSION: This study demonstrated that miR-136-5p affected the expression of A20 in IL-17-stimulated astrocytes.

HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling.

In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms. While prostratin strongly stimulates HMBA-induced HIV-1 transcription via improved P-TEFb activation, HMBA is capable of boosting NF-κB-dependent transcription initiation by suppressing prostratin-induced expression of the deubiquitinase A20, a negative feedback regulator in the NF-κB signaling pathway. In addition, HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-κB inhibitor IκBα, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-κB, a prerequisite for stimulation of transcription initiation. Thus, by blocking the negative feedback circuit, HMBA functions as a signaling enhancer of the NF-κB signaling pathway.

A20 reduces lipid storage and inflammation in hypertrophic adipocytes via p38 and Akt signaling.

Adipose tissue plays a vital role in the development of obesity and related disorders. Our previous study showed that A20, an ubiquitin-editing enzyme with anti-inflammation function, attenuated free fatty acids (FFAs)-induced lipid accumulation in nonalcoholic steatohepatitis. Here, we investigated A20 expression in adipose tissue of obese individuals and its effects on 3T3-L1 lipogenesis as well as the likely mechanisms underlying this process. By re-annotation of raw microarray data downloaded from Gene Expression Omnibus, we found that obese individuals showed significantly higher A20 mRNA levels in adipocytes. In vitro, A20 inhibited MCP-1 and IL-6 secretion in adipocytes. Forced expression of A20 resulted in decreased expression of key markers of lipogenesis and adipogenesis, such as sterol regulatory element binding protein 1c (SREBP-1c) and adipogenesis (aP2), leading to less lipids accumulation in differentiated 3T3-L1 cells. This process was concomitant with attenuated activation of p38 and Akt signaling. Our results suggest that A20 may have therapeutic potential for obesity and related diseases. The mechanisms involved the suppression of lipid storage and inflammation in adipocytes.

The Discovery and Validation of Biomarkers for the Diagnosis of Esophageal Squamous Dysplasia and Squamous Cell Carcinoma.

The 5-year survival rate of esophageal cancer is less than 10% in developing countries, where more than 90% of these cancers are esophageal squamous cell carcinomas (ESCC). Endoscopic screening is undertaken in high incidence areas. Biomarker analysis could reduce the subjectivity associated with histologic assessment of dysplasia and thus improve diagnostic accuracy. The aims of this study were therefore to identify biomarkers for esophageal squamous dysplasia and carcinoma. A publicly available dataset was used to identify genes with differential expression in ESCC compared with normal esophagus. Each gene was ranked by a support vector machine separation score. Expression profiles were examined, before validation by qPCR and IHC. We found that 800 genes were overexpressed in ESCC compared with normal esophagus (P < 10(-5)). Of the top 50 genes, 33 were expressed in ESCC epithelium and not in normal esophagus epithelium or stroma using the Protein Atlas website. These were taken to qPCR validation, and 20 genes were significantly overexpressed in ESCC compared with normal esophagus (P < 0.05). TNFAIP3 and CHN1 showed differential expression with IHC. TNFAIP3 expression increased gradually through normal esophagus, mild, moderate and severe dysplasia, and SCC (P < 0.0001). CHN1 staining was rarely present in the top third of normal esophagus epithelium and extended progressively towards the surface in mild, moderate, and severe dysplasia, and SCC (P < 0.0001). Two novel promising biomarkers for ESCC were identified, TNFAIP3 and CHN1. CHN1 and TNFAIP3 may improve diagnostic accuracy of screening methods for ESCC. Cancer Prev Res; 9(7); 558-66. ©2016 AACR.