Details

Autor(en) / Beteiligte

• Wilkinson, Melissa L.
• Gou, Changjiang
• Stevenson, Emily R.
• Abramova, Elena
• Freeman, Bruce A.
• Gow, Andrew J.

Titel

Mechanisms of Nitroalkene Inhibition of TLR4 Mediated Macrophage Activation

Ist Teil von

• The FASEB journal, 2022-05, Vol.36 (S1), p.n/a

Ort / Verlag

United States: The Federation of American Societies for Experimental Biology

Erscheinungsjahr

2022

Quelle

Wiley

Beschreibungen/Notizen

• Fatty acid nitroalkenes, such as nitro‐oleic acid (OA‐NO2), 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‐NO2 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‐NO2 administration are through inhibition of TLR4 responses specifically through NF‐κB inhibition. To investigate this hypothesis, OA‐NO2 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‐NO2 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‐NO2 also inhibited iNOS protein expression when compared to LPS (443±52.9* vs 88±5.5†% density). To examine OA‐NO2 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‐NO2 stabilization of NF‐κB inhibitory proteins Iκ‐B protein levels were assessed. Iκ‐B was not stabilized, as protein concentrations were decreased with OA‐NO2 regardless of LPS stimulation. This indicated OA‐NO2 may directly bind to NF‐κB, preventing its activation and marking Iκ‐B for degradation. To test this hypothesis, an alkyne‐containing OA‐NO2 derivative was used to identify proteins that are adducted by OA‐NO2. After reaction with biotinylated azide, streptavidin was used to capture alkynyl‐OA‐NO2 adducted proteins. This revealed that OA‐NO2 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‐NO2 inhibits TLR4 mediated pro‐inflammatory gene expression responses through direct post‐translational modification and inhibition of NF‐κB. OA‐NO2 has the potential to alter other thiol containing proteins, meaning it may also act as a metabolic regulator. Our early data suggests that OA‐NO2 reduces LPS stimulation of glycolysis mediated acidification (6±1.1* vs 2±0.3†mpH/min). OA‐NO2 also reduces mitochondrial respiration regardless of LPS activation (9±1.5* 3±0.6*; 1±0.2#†pmol/min). Therefore, OA‐NO2 may inhibit macrophage activation through metabolic inhibition. In summary, OA‐NO2 is a potent regulator of pro‐inflammatory macrophage signaling and metabolism, indicating it potential therapeutic use in acute lung injury.