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Mechanisms of Nitroalkene Inhibition of TLR4 Mediated Macrophage Activation
Author(s) -
Wilkinson Melissa L.,
Gou Changjiang,
Stevenson Emily R.,
Abramova Elena,
Freeman Bruce A.,
Gow Andrew J.
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r6056
Subject(s) - tlr4 , chemistry , alveolar macrophage , gene expression , signal transduction , macrophage , pharmacology , receptor , microbiology and biotechnology , biochemistry , in vitro , biology , gene
Fatty acid nitroalkenes, such as nitro‐oleic acid (OA‐NO 2 ), are endogenously formed signaling mediators. They have demonstrated anti‐inflammatory properties but have not been extensively studied in the lung. We have previously shown that intratracheal administration of OA‐NO 2 reduces bleomycin‐mediated acute lung injury histopathologically and impacts pulmonary macrophage populations and activation. TLR4 is the principal damage receptor in acute lung injury and its activation results in pro‐inflammatory signaling. We hypothesize that changes to macrophage phenotype with OA‐NO 2 administration are through inhibition of TLR4 responses specifically through NF‐κB inhibition. To investigate this hypothesis, OA‐NO 2 effects on macrophage activation were examined in LPS‐activated RAW 267.4 cells. Following LPS treatment cells were collected either 6h, for gene expression analysis, or 24h for protein and metabolic analysis. qPCR showed OA‐NO 2 inhibited TLR4 target gene expression IL‐6 , PDGS2, and NOS2 in response to LPS (51±9.3* vs 19±12.3 † ; 45±15.0* vs 6±1.0 † ; 30±7.3* vs 1.4±0.8 † ‐fold change, respectively). OA‐NO 2 also inhibited iNOS protein expression when compared to LPS (443±52.9* vs 88±5.5 † % density). To examine OA‐NO 2 effects on transcription factor function, we tested its effects on LPS‐mediated increases in NF‐κB activity in RAW Blue cells, where there was a reduction in SEAP activity (10±0.7* vs 4±0.1 † rate). To determine if reduced activity could be attributed to OA‐NO 2 stabilization of NF‐κB inhibitory proteins Iκ‐B protein levels were assessed. Iκ‐B was not stabilized, as protein concentrations were decreased with OA‐NO 2 regardless of LPS stimulation. This indicated OA‐NO 2 may directly bind to NF‐κB, preventing its activation and marking Iκ‐B for degradation. To test this hypothesis, an alkyne‐containing OA‐NO 2 derivative was used to identify proteins that are adducted by OA‐NO 2 . After reaction with biotinylated azide, streptavidin was used to capture alkynyl‐OA‐NO 2 adducted proteins. This revealed that OA‐NO 2 adducts both the p50 and p65 subunits of NF‐κB, a reaction that does not prevent nuclear translocation, as determined by NF‐κB subunit detection following nuclear and cytoplasmic fractioning but does prevents its ability to transcribe downstream targets. This demonstrates that OA‐NO 2 inhibits TLR4 mediated pro‐inflammatory gene expression responses through direct post‐translational modification and inhibition of NF‐κB. OA‐NO 2 has the potential to alter other thiol containing proteins, meaning it may also act as a metabolic regulator. Our early data suggests that OA‐NO 2 reduces LPS stimulation of glycolysis mediated acidification (6±1.1* vs 2±0.3 † mpH/min). OA‐NO 2 also reduces mitochondrial respiration regardless of LPS activation (9±1.5* 3±0.6*; 1±0.2 #† pmol/min). Therefore, OA‐NO 2 may inhibit macrophage activation through metabolic inhibition. In summary, OA‐NO 2 is a potent regulator of pro‐inflammatory macrophage signaling and metabolism, indicating it potential therapeutic use in acute lung injury.