Oxidized Phosphatidylcholines Induce Map Kinase-dependent Production of Inflammatory Mediators in Human Airway Fibroblasts
Bibliographic record
Abstract
Abstract RATIONALE: Oxidative stress in asthma pathobiology generates bioactive oxidized phosphatidylcholines, most prominently OxPAPC. OxPAPC activates intracellular kinases, transient receptor potential ankyrin 1 (TRPA1)-mediated Ca2+ influx, and biosynthesis inflammatory mediators by airway smooth muscle. Mechanisms remain unclear, and whether OxPAPC also “activate” airway fibroblasts that regulate local inflammation and tissue remodeling is unknown. Here, using human airway fibroblasts (HAF) we test whether OxPAPC induces inflammatory gene expression and release, and survey signaling pathways required for this response, including NFκB, TRPA1, and mitogen activated protein kinases (MAPK). METHODS: We used kBU NF-κB-BEAS 2B luciferase reporter cells to measure OxPAPC (10-80µg/mL, 18 hr) effects on NFkB-induced gene transcription. With low passage, primary cultured HAF from lung resection specimens we used qRT-PCR for GM-CSF, IL-8, cyclooxygenase 2 (COX2) and TRPA1 after OxPAPC exposure (80µg/mL, 24 hrs). With Luminex assays we measured GM-CSF, IL-8 and IL-6 release. Some cultures also received the TRPA1 antagonist, A967079 (1µM). Using immunoblotting, we monitored phosphorylation of p42/p44 MAPK (Thr202, Tyr204) and p38 MAPK (Thr180/Tyr182) after OxPAPC exposure. Some cultures also received inhibitors of p42/p44 or p38 MAPK (U0126 and SB202190, 10μM). We measured effects of MAPK inhibitors on OxPAPC-induced Ca2+ influx in Fluo4-loaded HAF using a Cytation 5 reader. Statistical significance was tested by one-way ANOVA and Tukey's post hoc test, N ≥ 5). RESULTS: OxPAPC did not induce NF-κB dependent transcription in luciferase reporter cells. In HAF, OxPAPC increased GM-CSF (3-fold), IL-8 (9-fold) and COX2 (8-fold) mRNA, but not TRPA1. OxPAPC induced a 3-fold and 6-fold increase in IL-6 and IL-8 release, respectively. TRPA1 inhibition decreased OxPAPC induction of GM-CSF, IL-8 and COX2 mRNA by 70-85%, but did not affect the release of IL-6 and IL-8 protein. Within 30 minutes, OxPAPC induced a significant 6-fold increase in phospho-p42/p44 and p38 MAPK that decreased by ∼50% after 2 hrs, but was sustained to 4 hours. TRPA1 inhibition had little effect on OxPAPC-triggered MAPK phosphorylation. However, qRT-PCR indicated that inhibiting p42/p44 and p38 MAPK abrogated OxPAPC induction of GM-CSF and IL-8 mRNA. Inhibition of p42/p44 or p38 MAPK supressed OxPAPC-induced Ca2+ flux by 25-29%. CONCLUSIONS: OxPAPC induce inflammatory gene expression and release in HAF by triggering integrated signaling pathways involving TRPA1 and MAPKs. These data further support a role for OxPAPC as a mediator of oxidative stress that promotes the contributions of airway structural cells such as HAF to airway inflammation and remodeling
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".