Vaccinia virus BTB-Kelch proteins C2 and F3 inhibit NF-κB activation.

Vaccinia virus (VACV) encodes scores of proteins that suppress host innate immunity and many of these target intracellular signalling pathways leading to activation of inflammation. The transcription factor NF-κB plays a critical role in the host response to infection and is targeted by many viruses, including VACV that encodes 12 NF-κB inhibitors that interfere at different stages in this signalling pathway. Here we report that VACV proteins C2 and F3 are additional inhibitors of this pathway. C2 and F3 are BTB-Kelch proteins that are expressed early during infection, are non-essential for virus replication, but affect the outcome of infection in vivo. Using reporter gene assays, RT-qPCR analyses of endogenous gene expression, and ELISA, these BTB-Kelch proteins are shown here to diminish NF-κB activation by reducing translocation of p65 into the nucleus. C2 and F3 are the 13th and 14th NF-κB inhibitors encoded by VACV. Remarkably, in every case tested, these individual proteins affect virulence in vivo and therefore have non-redundant functions. Lastly, immunisation with a VACV strain lacking C2 induced a stronger CD8+ T cell response and better protection against virus challenge.

Vaccinia Virus BBK E3 Ligase Adaptor A55 Targets Importin-Dependent NF-κB Activation and Inhibits CD8+ T-Cell Memory.

Viral infection of cells is sensed by pathogen recognition receptors that trigger an antiviral innate immune response, and consequently viruses have evolved countermeasures. Vaccinia virus (VACV) evades the host immune response by expressing scores of immunomodulatory proteins. One family of VACV proteins are the BTB-BACK (broad-complex, tram-trac, and bric-a-brac [BTB] and C-terminal Kelch [BACK]) domain-containing, Kelch-like (BBK) family of predicted cullin-3 E3 ligase adaptors: A55, C2, and F3. Previous studies demonstrated that gene A55R encodes a protein that is nonessential for VACV replication yet affects viral virulence in vivo Here, we report that A55 is an NF-κB inhibitor acting downstream of IκBα degradation, preventing gene transcription and cytokine secretion in response to cytokine stimulation. A55 targets the host importin α1 (KPNA2), acting to reduce p65 binding and its nuclear translocation. Interestingly, while A55 was confirmed to coprecipitate with cullin-3 in a BTB-dependent manner, its NF-κB inhibitory activity mapped to the Kelch domain, which alone is sufficient to coprecipitate with KPNA2 and inhibit NF-κB signaling. Intradermal infection of mice with a virus lacking A55R (vΔA55) increased VACV-specific CD8+ T-cell proliferation, activation, and cytotoxicity in comparison to levels of the wild-type (WT) virus. Furthermore, immunization with vΔA55 induced increased protection to intranasal VACV challenge compared to the level with control viruses. In summary, this report describes the first target of a poxvirus-encoded BBK protein and a novel mechanism for DNA virus immune evasion, resulting in increased CD8+ T-cell memory and a more immunogenic vaccine.IMPORTANCE NF-κB is a critical transcription factor in the innate immune response to infection and in shaping adaptive immunity. The identification of host and virus proteins that modulate the induction of immunological memory is important for improving virus-based vaccine design and efficacy. In viruses, the expression of BTB-BACK Kelch-like (BBK) proteins is restricted to poxviruses and conserved within them, indicating the importance of these proteins for these medically important viruses. Using vaccinia virus (VACV), the smallpox vaccine, we report that the VACV BBK protein A55 dysregulates NF-κB signaling by disrupting the p65-importin interaction, thus preventing NF-κB translocation and blocking NF-κB-dependent gene transcription. Infection with VACV lacking A55 induces increased VACV-specific CD8+ T-cell memory and better protection against VACV challenge. Studying viral immunomodulators therefore expands not only our understanding of viral pathogenesis and immune evasion strategies but also of the immune signaling cascades controlling antiviral immunity and the development of immune memory.

Hydrogen-rich water protects against acetaminophen-induced hepatotoxicity in mice.

AIM: To investigate the hepatoprotective effects and mechanisms of hydrogen-rich water (HRW) in acetaminophen (APAP)-induced liver injury in mice. METHODS: Male mice were randomly divided into the following four groups: normal saline (NS) control group, mice received equivalent volumes of NS intraperitoneally (ip); HRW control group, mice were given HRW (same volume as the NS group); APAP + NS group, mice received NS ip for 3 d (5 mL/kg body weight, twice a day at 8 am and 5 pm) after APAP injection; APAP + HRW group, mice received HRW for 3 d (same as NS treatment) after APAP challenge. In the first experiment, mice were injected ip with a lethal dose of 750 mg/kg APAP to determine the 5-d survival rates. In the second experiment, mice were injected ip with a sub-lethal dose of 500 mg/kg. Blood and liver samples were collected at 24, 48, and 72 h after APAP injection to determine the degree of liver injury. RESULTS: Treatment with HRW resulted in a significant increase in the 5-d survival rate compared with the APAP + NS treatment group (60% vs 26.67%, P < 0.05). HRW could significantly decrease the serum alanine aminotransferase level (24 h: 4442 ± 714.3 U/L vs 6909 ± 304.8 U/L, P < 0.01; 48 h: 3782 ± 557.5 U/L vs 5111 ± 404 U/L, P < 0.01; and 3255 ± 337.4 U/L vs 3814 ± 250.2 U/L, P < 0.05, respectively) and aspartate aminotransferase level (24 h: 4683 ± 443.4 U/L vs 5307 ± 408.4 U/L, P < 0.05; 48 h: 3392 ± 377.6 U/L vs 4458 ± 423.6 U/L, P < 0.01; and 3354 ± 399.4 U/L vs 3778 ± 358 U/L, respectively) compared with the APAP treatment group. The alkaline phosphatase, total bilirubin and lactate dehydrogenase levels had the same result. Seventy-two hours after APAP administration, liver samples were collected for pathological examination and serum was collected to detect the cytokine levels. The liver index (5.16% ± 0.26% vs 5.88% ± 0.073%, P < 0.05) and percentage of liver necrosis area (27.73% ± 0.58% vs 36.87% ± 0.49%, P < 0.01) were significantly lower in the HRW-treated animals. The malonyldialdehyde (MDA) contents were significantly reduced in the HRW pretreatment group, but they were increased in the APAP-treated group (10.44 ± 1.339 nmol/mg protein vs 16.70 ± 1.646 nmol/mg protein, P < 0.05). A decrease in superoxide dismutase (SOD) activity in the APAP treatment group and an increase of SOD in the HRW treatment group were also detected (9.74 ± 0.46 U/mg protein vs 12.1 ± 0.67 U/mg protein, P < 0.05). Furthermore, HRW could significantly increase the glutathione (GSH) contents (878.7 ± 76.73 mg/g protein vs 499.2 ± 48.87 mg/g protein) compared with the APAP treatment group. Meanwhile, HRW could reduce the inflammation level (serum TNF-α: 399.3 ± 45.50 pg/L vs 542.8 ± 22.38 pg/L, P < 0.05; and serum IL-6: 1056 ± 77.01 pg/L vs 1565 ± 42.11 pg/L, P < 0.01, respectively). In addition, HRW could inhibit 4-HNE, nitrotyrosine formation, JNK phosphorylation, connexin 32 and cytochrome P4502E expression. Simultaneously, HRW could facilitate hepatocyte mitosis to promote liver regeneration. CONCLUSION: HRW has significant therapeutic potential in APAP-induced hepatotoxicity by inhibiting oxidative stress and inflammation and promoting liver regeneration.

FoxM1 overexpression promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma.

AIM: To investigate the expression of forkhead box protein M1 (FoxM1) in the process of epithelial mesenchymal transition in hepatocellular carcinoma (HCC) and its role in metastasis. METHODS: FoxM1 and E-cadherin expression in HCC tissue microarray specimens was evaluated by immunohistochemical staining, and statistical methods were applied to analyze the correlation between FoxM1 and epithelial-mesenchymal transition (EMT). Kaplan-Meier analysis of the correlation between the FoxM1 expression level and recurrence or overall survival of HCC patients was performed. The expression of FoxM1, E-cadherin and snail homologue 1 (SNAI1) in HCC cell lines was evaluated by real-time reverse transcription-polymerase chain reaction and Western blot. Hepatocyte growth factor (HGF) was used to induce EMT and stimulate cell migration in HCC cells. The expression of FoxM1 and SNAI1 was regulated by transfection with plasmids pcDNA3.1 and siRNAs in vitro. The occurrence of EMT was evaluated by Transwell assay, morphologic analysis and detection of the expression of EMT markers (E-cadherin and vimentin). Luciferase and chromatin immunoprecipitation assays were used to evaluate whether SNAI1 is a direct transcriptional target of FoxM1. RESULTS: FoxM1 expression was increased significantly in HCC compared with para-carcinoma (10.7 ± 0.9 vs 8.2 ± 0.7, P < 0.05) and normal hepatic (10.7 ± 0.9 vs 2.7 ± 0.4, P < 0.05) tissues. Overexpression of FoxM1 was correlated with HCC tumor size, tumor number, macrovascular invasion and higher TNM stage, but was negatively correlated with E-cadherin expression in microarray specimens and in cell lines. FoxM1 overexpression was correlated significantly with HCC metastasis and EMT. In vitro, we found that FoxM1 plays a key role in HGF-induced EMT, and overexpression of FoxM1 could suppress E-cadherin expression and induce EMT changes, which were associated with increased HCC cell invasiveness. Next, we confirmed that FOXM1 directly binds to and activates the SNAI1 promoter, and we identified SNAI1 as a direct transcriptional target of FOXM1. Moreover, inhibiting the expression of SNAI1 significantly inhibited FoxM1-mediated EMT. CONCLUSION: FoxM1 overexpression promotes EMT and metastasis of HCC, and SNAI1 plays a critical role in FoxM1-mediated EMT.