A20 Expression Research Articles

Chronic ingestion of alcohol modulates expression of ubiquitin editing enzyme A20 in lung macrophages

Background: Alcohol abuse is involved in the pathogenesis of multiple organ disorders; the underlying mechanism is incompletely understood. The ubiquitin editing enzyme A20 is involved in regulating activities in the cell. Suppression of A20 is suggested as one factor in the initiation of inflammation. This study investigates the mechanism by which chronic alcohol consumption modulates the levels of ubiquitin editing enzyme A20 in macrophages and further contributes to induce endothelial barrier dysfunction in the lung.
 Methods: Mice were gavage-fed with 40% alcohol daily for 0- 3 weeks. Airway macrophages were collected by lung lavage. Expression of ubiquitin editing enzyme A20 in isolated macrophages was assessed at both mRNA and protein levels. The endothelial barrier function of the lung was evaluated by the Evans blue method.
 Results: Mice treated with alcohol for 3 weeks showed an increase in cell infiltration in the lung in response to exposure to peptidoglycan; over 80% of the infiltrated cells were macrophages. Furthermore, we observed that A20 level was suppressed in macrophages of mice treated with alcohol; the levels of tumor necrosis factor, interleukin-6 and nuclear factor kappa B in macrophage were increased. In addition, the endothelial barrier function of the lung was compromised, showing excessive infiltration of Evans blue in the lung indicating lung edema. Pretreatment with synthesized A20 inhibited alcohol-induced lung endothelial barrier dysfunction.
 Conclusions: We conclude that chronic alcohol ingestion disturbs the endothelial barrier function in the lung by modulating macrophage properties. Increase in A20 in the cell may have potential for the treatment of inflammatory disorders.

Role of A20/TNFAIP3 deficiency in lupus nephritis in MRL/lpr mice.

The activation of the nuclear factor-κB (NF-κB) signaling pathway gives rise to inflammation in the pathogenesis of lupus nephritis (LN), with A20 serving as a negative feedback regulator and ubiquitin C‑terminal hydrolase L1 (UCH-L1) acting as a downstream target protein. However, their roles in the mechanism of LN remain undetermined. In the present study, the expression of A20 and UCH-L1, the activity of NF-κB and ubiquitin-proteasome system (UPS) were measured in MRL/lpr mice and A20 gene silenced podocytes. The severity of podocyte injury and immune complex deposits were detected by transmission electron microscopy. The in vivo experiments revealed that A20 failed to terminate the activation of NF-κB, which was accompanied by UCH-L1 overexpression, ubiquitin accumulation, and glomerular injury in LN mice. Immunosuppression therapy did improve LN progression by attenuating A20 deficiency. In vitro experiments confirmed that tumor necrosis factor-α induced NF-κB activation, which led to UCH-L1 overexpression, UPS impairment, the upregulation of desmin and the downregulation of synaptopodin in A20 gene silenced podocytes. Thus, the results of the present study suggest that A20 regulates UCH-L1 expression via the NF-κB signaling pathway and A20 deficiency might play an important role in LN pathogenesis. Therefore, the A20 protein may serve as a promising therapeutic target for LN.

The ubiquitin-editing enzyme A20 regulates synapse remodeling and efficacy

Ubiquitination and its reverse process, deubiquitination, play essential roles in neural development, function, and plasticity. A20, a ubiquitin editing enzyme that can remove K63-polyubiquitin chains from substrates and attach K48-polyubiquitin chains to them, is a critical component in the NF-κB signaling pathway in the immune system. This dual ubiquitin enzyme is also present in mammalian brains, but its potential role in neurons and synapses is unknown. We show that A20 in pyramidal neurons potently regulates dendritic arborization, spine morphogenesis, and synaptic transmission through an NF-κB-dependent mechanism. In cultured hippocampal neurons, overexpression of A20 reduced dendritic complexity and spine size and density, whereas A20 knockdown increased spine size and density, as well as clustering of the postsynaptic scaffold PSD-95 and glutamate receptor subunit GluA1. A20 effects in vitro were recapitulated in vivo where increasing or decreasing A20 expression in mouse brains reduced and enhanced spine density, respectively. Functionally, A20 knockdown significantly increased the amplitude, but not frequency of miniature excitatory postsynaptic currents, suggesting a role in postsynaptic efficacy. A20 negatively regulated NF-κB activation in neurons and A20 mutants deficient in either the deubiquitinase or the ubiquitin ligase activity failed to suppress NF-κB activation or reduce spine morphogenesis. Finally, selective inhibition of NF-κB abolished A20 knockdown-elicited spine formation, suggesting that A20 exerts its modulation on synapses through NF-κB signaling. Together, our study reveals a previously unknown role for A20, the only known ubiquitin editing enzyme with both deubiquitinase and ubiquitin ligase activity, in dendritic arborization, spine remodeling, and synaptic plasticity.

273P - MALT1- A20 and NF-κB expression pattern in patients with non-Hodgkin lymphomas

BackgroundThe nuclear factor-κB (NF-κB) pathway has been considered an essential and tightly regulated signaling cascade that mediates the development, activation, and survival of lymphocytes for regulated immune responses. Moreover, many of the oncogenic mediators involved in the pathology of lymphoma are regulated by NF-κB. Abnormal NF-κB activation occurs during many pathological conditions. A20 (intracellular ubiquitin-editing protein) is a negative feedback regulator of NF-κB signaling. A20 was identified as a MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) substrate, and MALT1 can cleave A20 to impair its NF-κB inhibitory function. MethodsThis study was carried out on 100 subjects classified into the following groups: Group I: included 80 patients with Non-Hodgkin Lymphoma (NHL). Group II: Included 20 ages and sex matched healthy individuals as a control group. All participants were subjected to full history taking and clinical examination. All NHL cases were subjected to abdominal ultrasound & CT-scan for abdomen and metastatic work up: chest x-ray and bone scan. Blood sample were taken for CBC, ESR, Serum LDH and β2 microglobulin (β2M) levels and detection of MALT1, A20 and NF-κB genes expression in cDNA samples extracted from RNA samples by reverse transcription by using real time PCR using SYBR Green technique. ResultsThere was significant statistical increased expression of MALT1 and NF-κB and significant statistical decreased expression of A20 in NHL cases when compared with control. In NHL patients there was significant statistical decreased expression of MALT1, A20 and NF-κBin (HCV +ve) NHL cases when compared with controls and there was significant statistical increased expression of MALT1 and NF-κBand significant statistical decreased expression of A20 in (HCV-ve) NHL cases when compared with controls. ConclusionsMALT1- A20 and NF-κB have specific expression pattern in patients with Non Hodgkin Lymphomas. Legal entity responsible for the studyMenoufia university. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

A20 as an immune tolerance factor can determine islet transplant outcomes.

Islet transplantation can restore lost glycemic control in type 1 diabetes subjects but is restricted in its clinical application by a limiting supply of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signaling thresholds. Here, we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for ~45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon Tregs and was antigen specific, and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signaling, graft hyperinflammation, and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators, with no impact on glucose-stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signaling thresholds to favor immune tolerance and promotes islet allogeneic survival. Clinically, this would allow for reduced immunosuppression and support the use of alternate islet sources.

A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms

The cytokine TNF promotes inflammation either directly by activating the MAPK and NF-κB signaling pathways, or indirectly by triggering cell death. A20 is a potent anti-inflammatory molecule, and mutations in the gene encoding A20 are associated with a wide panel of inflammatory pathologies, both in human and in the mouse. Binding of TNF to TNFR1 triggers the NF-κB-dependent expression of A20 as part of a negative feedback mechanism preventing sustained NF-κB activation. Apart from acting as an NF-κB inhibitor, A20 is also well-known for its ability to counteract the cytotoxic potential of TNF. However, the mechanism by which A20 mediates this function and the exact cell death modality that it represses have remained incompletely understood. In the present study, we provide in vitro and in vivo evidences that deletion of A20 induces RIPK1 kinase-dependent and -independent apoptosis upon single TNF stimulation. We show that constitutively expressed A20 is recruited to TNFR1 signaling complex (Complex I) via its seventh zinc finger (ZF7) domain, in a cIAP1/2-dependent manner, within minutes after TNF sensing. We demonstrate that Complex I-recruited A20 protects cells from apoptosis by stabilizing the linear (M1) ubiquitin network associated to Complex I, a process independent of its E3 ubiquitin ligase and deubiquitylase (DUB) activities and which is counteracted by the DUB CYLD, both in vitro and in vivo. In absence of linear ubiquitylation, A20 is still recruited to Complex I via its ZF4 and ZF7 domains, but this time protects the cells from death by deploying its DUB activity. Together, our results therefore demonstrate two distinct molecular mechanisms by which constitutively expressed A20 protect cells from TNF-induced apoptosis.

IL-1α and IL-1β promote NOD2-induced immune responses by enhancing MAPK signaling

Both toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) induce a tightly regulated inflammatory response at risk of causing tissue damage, depending on the effectiveness of ensuing negative feedback regulatory mechanisms. Cross-regulation between TLRs, NLRs, and cytokine receptors has been observed. However, the cross-regulation between interleukin-1 (IL-1) receptors and NOD2 is not completely understood. In this study, we found that IL-1α/β increased NOD2-induced inflammatory response in human monocytic THP1 cells, peripheral blood mononuclear cells (PBMCs), mouse macrophage RWA264.7 cells and spleen cells, and in an in vivo experiment. IL-1α/β pre-treatment induced the production of CXC chemokines, including growth-regulated oncogene (GRO)-α, GRO-β, and IL-8, and proinflammatory cytokines, including IL-1β, IL-6, and TNFα, which are induced by the activation of NOD2, in a dose- and time-dependent manner. However, pre-treatment with the NOD2 ligand muramyl dipeptide (MDP) did not up-regulate the expression of cytokines induced by IL-1α/β re-treatment. IL-1β treatment increased the expression of A20, which is an important inhibitor of the innate immune response. However, the overexpression of A20 failed to inhibit MDP-induced cytokine production, suggesting that A20 had no effects on the NOD2-induced immune response. In addition, IL-1α/β increased the expression of NOD2 and its downstream adaptor RIP2, and IL-1α/β pre-treatment increased MDP-induced activation of mitogen-activated protein kinases (MAPKs), including ERK, JNK, and P38, which contributed to MDP-induced cytokine production. Based on these results, IL-1α/β promote the NOD2-induced immune responses by enhancing MDP-induced activation of MAPK signaling pathways.

Targeting Degradation of the Transcription Factor C/EBPβ Reduces Lung Fibrosis by Restoring Activity of the Ubiquitin-Editing Enzyme A20 in Macrophages

Although recent progress provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), rare anti-PF therapeutics show definitive promise for treating this disease. Repeated lung epithelial injury results in injury-repairing response and inflammation, which drive the development of PF. Here, we report that chronic lung injury inactivated the ubiquitin-editing enzyme A20, causing progressive accumulation of the transcription factor C/EBPβ in alveolar macrophages (AMs) from PF patients and mice, which upregulated a number of immunosuppressive and profibrotic factors promoting PF development. In response to chronic lung injury, elevated glycogen synthase kinase-3β (GSK-3β) interacted with and phosphorylated A20 to suppress C/EBPβ degradation. Ectopic expression of A20 or pharmacological restoration of A20 activity by disturbing the A20-GSK-3β interaction accelerated C/EBPβ degradation and showed potent therapeutic efficacy against experimental PF. Our study indicates that a regulatory mechanism of the GSK-3β-A20-C/EBPβ axis in AMs may be a potential target for treating PF and fibroproliferative lung diseases.

High A20 expression negatively impacts survival in patients with breast cancer.

BackgroundA20 protein has ubiquitin-editing activities and acts as a key regulator of inflammation and immunity. Previously, our group showed that A20 promotes tumor metastasis through multi-monoubiquitylation of SNAIL1 in basal-like breast cancer. Here, we investigated survival outcomes in patients with breast cancer according to A20 expression.Patients and methodsWe retrospectively collected tumor samples from patients with breast cancer. Immunohistochemistry (IHC) with an A20-specific antibody was performed, and survival outcomes were analyzed.ResultsA20 expression was evaluated in 442 patients. High A20 expression was associated with advanced anatomical stage and young age. High A20 expression showed significantly inferior recurrence-free-survival and overall-survival (P<0.001 and P<0.001, respectively). Multivariate analysis showed that A20 was an independent prognostic marker for RFS (HRs: 2.324, 95% CIs: 1.446–3.736) and OS (HRs: 2.629, 95% CIs: 1.585–4.361). In human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) subtypes, high A20 levels were associated with poor OS.ConclusionWe found that A20 expression is a poor prognostic marker in breast cancer. The prognostic impact of A20 was pronounced in aggressive tumors, such as HER2-positive and TNBC subtypes. Our findings suggested that A20 may be a valuable target in patients with aggressive breast cancer.

Inhibition of transmembrane TNF-\u03b1 shedding by a specific antibody protects against septic shock

Transmembrane TNF-α (tmTNF-α) and secretory TNF-α (sTNF-α) display opposite effects in septic shock. Reducing tmTNF-α shedding can offset the detrimental effects of sTNF-α and increase the beneficial effect of tmTNF-α. We previously developed a monoclonal antibody that is specific for tmTNF-α and does not cross-react with sTNF-α. In this study, we show that this antibody can specifically suppress tmTNF-α shedding by competing with a TNF-α converting enzyme that cleaves the tmTNF-α ectodomain to release sTNF-α. This tmTNF-α antibody significantly inhibited LPS-induced secretion of interleukin (IL)-1β, IL-6, interferon-β, and nitric oxide by monocytes/macrophages, and protected mice from septic shock induced by lipopolysaccharide (LPS) or cecal ligation and puncture, while reducing the bacterial load. The mechanism associated with the protective effect of this tmTNF-α antibody involved promotion of LPS-induced toll-like receptor 4 (TLR4) internalization and degradation by recruiting Triad3A to TLR4. Moreover, the tmTNF-α antibody inhibited LPS-induced activation of nuclear factor-κB and interferon regulatory factor 3 pathways by upregulating expression of A20 and monocyte chemotactic protein-induced protein 1. Similarly, treatment of macrophages with exogenous tmTNF-α suppressed LPS/TLR4 signaling and release of proinflammatory cytokines, indicating that increased levels of tmTNF-α promoted by the antibody contributed to its inhibitory effect. Thus, use of this tmTNF-α antibody for specific suppression of tmTNF-α shedding may be a promising strategy to treat septic shock.

Biological Properties of Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells

To obtain induced pluripatent stem cells (iPSC) from peripheral blood mononucleated cells and further induce differentiation into mesenchymal stem cells (MSC), and to compare the biological characteristics of iPSC-derived MSC and other-derived MSC. Peripheral blood mononucleated cells were obtained and transduced with reprogramming factors by sendai virus vector. Induced differentiation of MSC was performed in 1 strain of iPSC that completed all identification, and their cell morphology and immunophenotype were identified by immu-nohistochemistry and flow cytometry. Adipogenic and osteogenic media were used to induce the adipogenic and osteogenic differentiation. The expression of immune-related transcription factors was identified by PCR to systematically elucidate the biological characteristics of iPSC-induced MSC. After transfection with sendai virus with reprogramming factor, the fate of peripheral blood mononucleated cells was reversed, initiating the expression of stem cell characteristics, the iPSC was successfully cloned, amplified, and purified, and finally the stable proliferation of iPSC was obtained. Mesenchymal stem cells derived from iPSC, had morphology consistent with other-derived MSC, and the immunophenotypes met the standard. iPSC-MSC possessed the ability of lipogenic and osteogenic differentiation. RT-PCR showed that iPSC-MSC was high expression to PDL1, and low expression to A20; besides, the expression level of STAT3 was equal to BM-MSC; and also as to the expression level of HIF1α and UC-MSC, which was lower than BM-MSC. Peripheral blood mononucleated cells successfully initiated the expression of stem cell characteristics after the transduction of sendai virus vector with reprogramming factors, and obtained multi-competent iPSC. iPSC can successfully be induced to the differentiation of MSC, and the iPSC-MSC have standard cell morphology, immunophenotype and differentiation ability. High expression of PDL1 and low expression of A20 in iPSC-MSC suggest that iPSC-derived cells have different biological characteristics in cell proliferation and immune regulation.

The expression of microRNA 146a in patients with ischemic stroke: an observational study.

AimWe conducted the present prospective study to assess the level of microRNA (miRNA) 146a in patients with ischemic stroke and its correlation with patients’ characteristics.MethodsWe conducted an observational study that included adult patients (≥18 years old) who presented within 24 hrs after the onset of the symptoms of acute ischemic stroke. In addition, age- and sex-matched healthy volunteers were included as control group. The primary outcome in the present study was the difference in miRNA 146a expression between patients with ischemic stroke and control group participants. The expression of miRNA 146a was measured using quantitative real-time PCR. Quantitative real-time PCR amplification and analysis were performed using Rotor-Gene Q thermal cycler.ResultsThe present study included 44 patients with ischemic stroke and 22 matched controls. Regarding the primary outcome of the present study, the median expression of miRNA 146a in patients with ischemic stroke was −1.98 fold (IQR −27.1–3.9) compared to 1.75 fold (IQR −2.25–5.27) in control group (P<0.001). However, the subgroup analysis showed that the expression of miRNA 146a was significantly downregulated in comatosed patients only (P<0.001). The expression of miRNA 146a correlated negatively with Glasgow Coma Scale score in comatose patients (r=−0.352, P=0.022).ConclusionIn conclusion, the expression of miRNA 146a is significantly downregulated in ischemic stroke patients. Further studies are needed to assess its diagnostic utility and therapeutic potentials.

Hepatitis C Virus Induces the Ubiquitin-Editing Enzyme A20 via Depletion of the Transcription Factor Upstream Stimulatory Factor 1 To Support Its Replication.

Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20, is a ubiquitin-editing enzyme capable of ubiquitination or deubiquitination of its target proteins. In this study, we show that hepatitis C virus (HCV) infection could induce the expression of A20 via the activation of the A20 promoter. The induction of A20 by HCV coincided with the loss of upstream stimulatory factor 1 (USF-1), a transcription factor known to suppress the A20 promoter. The role of USF-1 in the regulation of the A20 promoter in HCV-infected cells was confirmed by the chromatin immunoprecipitation (ChIP) assay, and its depletion was apparently mediated by proteasomes, as USF-1 could be stabilized by the proteasome inhibitor MG132 to suppress the A20 expression. As the overexpression of A20 enhanced the replication of HCV and the silencing of A20 had the opposite effect, A20 is a positive regulator of HCV replication. Our further studies indicated that A20 enhanced the activity of the HCV internal ribosome entry site (IRES). In conclusion, our results demonstrated that HCV could induce the expression of A20 via the depletion of USF-1 to enhance its replication. Our study provided important information for further understanding the interaction between HCV and its host cells.IMPORTANCE Hepatitis C virus establishes chronic infection in approximately 85% of the patients whom it infects. However, the mechanism of how HCV evades host immunity to establish persistence is unclear. In this report, we demonstrate that HCV could induce the expression of the ubiquitin-editing enzyme A20, an important negative regulator of the tumor necrosis factor alpha (TNF-α) and NF-κB signaling pathways. This induction of A20 enhanced HCV replication as it could stimulate the HCV IRES activity to enhance the translation of HCV proteins. The induction of A20 was mediated by the depletion of USF-1, a suppressor of the A20 promoter. Our study thus provides important information for further understanding the interaction between HCV and its host cells.

Berberine improves intestinal epithelial tight junctions by upregulating A20 expression in IBS-D mice

To investigate effects of berberine exerts on A20 expression and regulation of intestinal epithelial tight junctions via the TNF-α-NF-κB-MLCK pathway in Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D).C57BL/6 wild type (WT) and A20 IEC-KO mice (48 each) were randomly divided into normal control (NC), model control (MC), rifaximin and berberine groups (12 mice per group). An experimental model of IBS-D was established using 4% acetic acid and evaluated by haematoxylin-eosin (HE) staining. rifaximin and berberine mice were treated with rifaximin and berberine, respectively.Intestinal epithelial space of WT berberine mice improved more than A20 IEC-KO berberine mice compared to MC mice. WT berberine mice exhibited greater expression of A20 compared with MC mice(P < 0.01). TNF-α, NF-kB p65, MLCK, MLC, TRAF6 and RIP1 levels in A20 IEC-KO and WT berberine mice were all decreased compared to MC mice(P all<0.05). NF-κB p65, MLCK and TRAF6 levels were increased in A20 IEC-KO berberine mice as compared to WT berberine mice (P all<0.05). Intestinal epithelial levels of occludin, claudin-1, ZO-1 and F-actin increased in all berberine mice (P all<0.01-0.05), while occludin, claudin-1, and ZO-1 levels were lower in A20 IEC-KO berberine mice(P < 0.05).Berberine downregulates abnormal activation of the TNF-α-NF-κB-MLCK pathway by upregulating expression of A20 in a mouse model of IBS-D, thereby protecting intestinal epithelial tight junctions and repairing the damage IBS-D causes to the intestinal epithelial barrier.

Herbs-partitioned moxibustion improves intestinal epithelial tight junctions by upregulating A20 expression in a mouse model of Crohn’s disease

BackgroundTo investigate effects moxibustion exerts on A20 expression and regulation of intestinal epithelial tight junctions via the TNF-α-NF-κB-MLCK pathway in Crohn's disease (CD). MethodsC57BL/6 wild type (WT) and A20IEC−KO mice (48 each) were randomly divided into normal control (NC), model control (MC), mesalazine (MESA) and herbs-partitioned moxibustion (HPM) groups (12 mice per group). An experimental model of CD was established using 2, 4, 6 trinitrobenzene sulfonic acid. MESA and HPM mice were treated with MESA and HPM (at Tianshu (ST25) and Qihai (CV6)), respectively. In HPM group, moxa cones (0.5 cm in diameter and 0.3 cm in height) made of refined mugwort floss were placed on herbal cakes (medicinal formula dispensing [radix] Aconiti praeparata, [cortex] Cinnamomi, etc.) at Tianshu (ST25) and Qihai (CV6) and ignited. The moxa cones were ignited, and two moxa cones were used for each treatment once daily for 10 days. In MESA group, mice were fed MESA, which was prepared at a proportion of 1:0.0026, twice daily for 10 days. ResultsIntestinal epithelial ultrastructure of WT HPM mice improved more than A20IEC−KO HPM mice compared to MC mice. WT HPM mice exhibited greater expression of A20 compared with MC mice (P < 0.01). TNF-α, NF-kB p65, MLCK, MLC, TRAF6 and RIP1 levels in A20IEC−KO and WT HPM mice were all decreased compared to MC mice (Pall < 0.01). NF-κB p65、MLCK and TRAF6 levels were increased in A20IEC−KO HPM mice as compared to WT HPM mice (Pall < 0.05). Intestinal epithelial levels of occludin, claudin-1, ZO-1 and F-actin increased in all HPM mice (Pall < 0.01–0.05), while occludin, claudin-1, and ZO-1 levels were lower in A20 IEC-KO HPM mice (P < 0.05, P < 0.01, P < 0.01). ConclusionHPM downregulates abnormal activation of the TNF-α-NF-κB-MLCK pathway by upregulating expression of A20 in a mouse model of CD, thereby protecting intestinal epithelial tight junctions and repairing the damage CD causes to the intestinal epithelial barrier.

A20 in dendritic cells restrains intestinal anti-bacterial peptide expression and preserves commensal homeostasis.

Microbial dysbiosis commonly occurs in patients with inflammatory bowel diseases (IBD). Exogenous causes of dysbiosis such as antibiotics and diet are well described, but host derived causes are understudied. A20 is a potent regulator of signals triggered by microbial pattern molecules, and A20 regulates susceptibility to intestinal inflammation in mice and in humans. We now report that mice lacking A20 expression in dendritic cells, A20FL/FL CD11c-Cre mice (or A20dDC mice), spontaneously develop colitogenic intestinal dysbiosis that is evident upon weaning and precedes the onset of colitis. Intestines from A20dDC mice express increased amounts of Reg3β and Reg3γ, but not Ang4. A20 deficient DCs promote gut microbiota perturbation in the absence of adaptive lymphocytes. Moreover, A20 deficient DCs directly induce expression of Reg3β and Reg3γ but not Ang 4 in normal intestinal epithelial cell enteroid cultures in the absence of other cell types. These findings reveal a pathophysiological pathway in which defective expression of an IBD susceptibility gene in DCs drives aberrant expression of anti-bacterial peptides and luminal dysbiosis that in turn confers host susceptibility to intestinal inflammation.

Decreased A20 expression on circulating CD56bright NK cells contributes to a worse disease status in patients with ankylosing spondylitis.

A20, a pivotal anti-inflammatory protein, preserves immune homeostasis and regulates prolonged inflammation. A previous study has shown that A20 expression levels are down-regulated in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS). However, the precise role of A20 in reducing autoimmune disorders needs to be further elucidated. In this study, A20 expression was found to be preferentially reduced on circulating CD56bright natural killer (NK) cells in patients with AS, and its level was negatively correlated with that of proinflammatory cytokines. Further investigation demonstrated that A20 reduces interferon (IFN)-γ and tumour necrosis factor (TNF)-α production in CD56bright NK cells after stimulation with monokines or phorbol myristate acetate (PMA)/ionomycin(P/I). Furthermore, CD56bright NK cells isolated from AS patients promote TNF-α secretion by autologous monocytes, and increasing the A20 expression level partially attenuates this process. More importantly, decreased A20 expression on circulating CD56bright NK cells is associated with worse disease status in patients with AS. Our findings reveal that A20 participates in the pathogenesis of AS by negatively regulating CD56bright NK cells and that its reduced expression contributes to a worsened disease status in patients with AS.

Alpha-1-Antitrypsin Enhances Primary Human Macrophage Immunity Against Non-tuberculous Mycobacteria.

Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection.Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria.Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion—given to individuals with AAT deficiency—to control an ex vivo Mycobacterium intracellulare infection.Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare–induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis.Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.

Melatonin Suppresses Microglial Necroptosis by Regulating Deubiquitinating Enzyme A20 After Intracerebral Hemorrhage.

Cell death is deeply involved in pathophysiology of brain injury after intracerebral hemorrhage (ICH). Necroptosis, one of the recently discovered forms of cell death, plays an important role in various diseases, including ICH. Previous studies have suggested that a considerable number of neurons undergoes necroptosis after ICH. However, necroptosis of microglia after ICH has not been reported to date. The present study demonstrated for the first time that necroptosis occurred in the microglia surrounding the hematoma after ICH in C57 mice, and melatonin, a hormone that is predominantly synthesized in and secreted from the pineal gland, exerted a neuroprotective effect by suppressing this process. When we further explored the potential underlying mechanism, we found that melatonin inhibits RIP3-mediated necroptosis by regulating the deubiquitinating enzyme A20 (also known as TNFAIP3) expression after ICH. In summary, we have demonstrated the role of microglial necroptosis in the pathogenesis of ICH. More importantly, A20 was identified as a novel target of melatonin, which opens perspectives for future research.

Herbs-partitioned moxibustion alleviates aberrant intestinal epithelial cell apoptosis by upregulating A20 expression in a mouse model of Crohn's disease.

BACKGROUNDA20 inhibits intestinal epithelial cell apoptosis in Crohn’s disease, and herbs-partitioned moxibustion (HPM) has been demonstrated to be an effective treatment for Crohn’s disease. However, the mechanism by which HPM reduces intestinal epithelial cell apoptosis in Crohn’s disease has not been thoroughly elucidated to date.AIMTo elucidate whether HPM exerts its effects by upregulating A20 to affect intestinal epithelial cell apoptosis in a Crohn’s disease mouse model.METHODSIn this study, mice with A20 deletion in intestinal epithelial cells (A20IEC-KO) were utilized to establish a Crohn’s disease mouse model with 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration, as well as wild-type mice. Mice were randomly divided into normal control (NC), model control (MC), mesalazine (MESA), and HPM groups. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and serum endotoxin and apoptosis of epithelial cells were evaluated by enzyme-linked immunosorbent assay and terminal dUTP nick-end labeling assay accordingly. The protein expression levels of A20 and tumor necrosis factor receptor 1 (TNFR1)-related signaling molecules were evaluated by Western blot, and co-expression of A20 and TNFR1-associated death domain (TRADD) and co-expression of A20 and receptor-interacting protein 1 (RIP1) were observed by double immunofluorescence staining.RESULTSThe intestinal epithelial barrier was noted to have an improvement in the HPM group of wild-type (WT) mice compared with that in A20IEC-KO mice. Compared with A20 IEC-KO HPM mice, serum endotoxin levels and apoptosis percentages were decreased (P < 0.01), A20 expression levels were increased (P < 0.01), and expression of TNFR1, TRADDD, and RIP1 was decreased in the HPM group of WT mice (PTNFR1 < 0.05, PTRADD < 0.01, PRIP1 < 0.01). Both of the co-expression of A20/TRADD and A20/RIP1 showed a predominantly yellow fluorescence in the HPM group of WT mice, while a predominantly red fluorescence was noted in the HPM group of A20IEC-KO mice.CONCLUSIONOur findings suggest that HPM in treating Crohn’s disease functions possibly via upregulation of the A20 expression level, resulting in downregulation of TNFR1, TRADD, and RIP1 to alleviate increased cell apoptosis in the intestinal epithelial barrier in Crohn's disease.