NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Amino acid positions near the active site determine the reduced activity of human ACOD1 compared to murine ACOD1.

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homo sapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer.

Addition of Fibroblast-Stromal Cell Markers to Immune Synovium Pathotypes Better Predicts Radiographic Progression at 1 Year in Active Rheumatoid Arthritis.

Objectives: This study aims to investigate if addition of fibroblast-stromal cell markers to a classification of synovial pathotypes improves their predictive value on clinical outcomes in rheumatoid arthritis (RA). Methods: Active RA patients with a knee needle synovial biopsy at baseline and finished 1-year follow-up were recruited from a real-world prospective cohort. Positive staining for CD20, CD38, CD3, CD68, CD31, and CD90 were scored semiquantitatively (0-4). The primary outcome was radiographic progression defined as a minimum increase of 0.5 units of the modified total Sharp score from baseline to 1 year. Results: Among 150 recruited RA patients, 123 (82%) had qualified synovial tissue. Higher scores of CD20+ B cells, sublining CD68+ macrophages, CD31+ endothelial cells, and CD90+ fibroblasts were associated with less decrease in disease activity and greater increase in radiographic progression. A new fibroblast-based classification of synovial pathotypes giving more priority to myeloid and stromal cells classified samples as myeloid-stromal (57.7%, 71/123), lymphoid (31.7%, 39/123), and paucicellular pathotypes (10.6%, 13/123). RA patients with myeloid-stromal pathotype showed the highest rate of radiographic progression (43.7% vs. 23.1% vs. 7.7%, p = 0.011), together with the lowest rate of Boolean remission at 3, 6, and 12 months. Baseline synovial myeloid-stromal pathotype independently predicted radiographic progression at 1 year (adjusted OR: 3.199, 95% confidence interval (95% CI): 1.278, 8.010). Similar results were obtained in a subgroup analysis of treatment-naive RA. Conclusions: This novel fibroblast-based myeloid-stromal pathotype could predict radiographic progression at 1 year in active RA patients which may contribute to the shift of therapeutic decision in RA.

[Effect of acupuncture technique of Tiaoxin Tongdu on learning-memory ability and expressions of hippocampal VEGF and Ang-1 in rats with vascular dementia].

OBJECTIVE: To observe the effect of acupuncture technique of Tiaoxin Tongdu on learning-memory ability and expressions of hippocampal vascular endothelial growth factor (VEGF) and angiogenin-1 (Ang-1) in rats with vascular dementia (VD), and to explore the mechanism of acupuncture technique of Tiaoxin Tongdu for VD. METHODS: A total of 24 male SD rats were randomly divided into a sham operation group, a model group, a medication group and an acupuncture group after Morris water maze test, 6 rats in each group. VD model was established by permanent ligation of bilateral common carotid arteries in the model group, the medication group and the acupuncture group. Treatment was given on the next day after successful modeling. The rats in the acupuncture group were treated with acupuncture at "Baihui" (GV 20), "Shenting" (GV 24), "Shuigou" (GV 26), "Dazhui" (GV 14), "Fengfu" (GV 16), "Mingmen" (GV 4), "Neiguan" (PC 6), "Daling" (PC 7) and "Laogong" (PC 8) for 30 min; the rats in the medication group were treated with nimodipine solution (0.0625 g/kg) by gavage, once a day, for 2 weeks. Morris water maze test was used to detect the behavior of rats before modeling, 2 weeks after modeling and after intervention; after intervention, the expressions of VEGF and Ang-1 protein in hippocampus were detected by Western blot. RESULTS: Compared with the sham operation group, the average escape latency of rats in the model group was prolonged (P<0.01), and the times of crossing the original platform were reduced (P<0.01). Compared with the model group, the average escape latency of rats in the medication group and acupuncture group was significantly shortened (P<0.01), and the times of crossing the original platform were increased (P<0.01, P<0.05). Compared with the sham operation group, the expressions of VEGF and Ang-1 protein in hippocampus in the model group were increased (P<0.05, P<0.01). Compared with the model group, the expressions of VEGF and Ang-1 protein in the hippocampus in the medication group and acupuncture group were significantly increased (P<0.01, P<0.05). CONCLUSION: The acupuncture technique of Tiaoxin Tongdu can significantly improve the learning and memory ability of VD rats, and its mechanism may be related to up-regulating the expressions of VEGF and Ang-1 protein in hippocampus and inducing angiogenesis.