Hypoxia-Induced NLRP3 Inflammasome Activation in a Cellular Model for Pulmonary Arterial Hypertension
Bibliographic record
Abstract
Abstract Rationale: Pulmonary arterial hypertension (PAH) is a severe disease marked by vascular remodeling and right heart failure, with limited treatments available. Studies suggest macrophage pyroptosis, driven by NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome activation, contributes to inflammation in PAH. While NLRP3 involvement in PAH has been established, the trigger for activation remains unknown. Hypoxia, commonly used in PAH models, has been linked to reactive oxygen species (ROS) production, which may activate NLRP3 and drive pyroptosis via gasdermin D (GSDMD) lipidation. We hypothesized that hypoxia-induced ROS enhances macrophage pyroptosis in PAH. Methods: Mouse bone marrow-derived macrophages (BMDMs) were cultured under normoxic or hypoxic conditions (2% O₂ for 24 hours) with or without lipopolysaccharide (LPS) and nigericin (Ng) to induce pyroptosis. Cell death was assessed via lactate dehydrogenase (LDH) release and Zombie Red staining. Extracellular IL-1β and high-mobility group box 1 (HMGB1) were measured as markers of inflammasome activation and pyroptosis. Expression of NLRP3 and IL-1β was analyzed by qPCR. MCC950 (NLRP3 inhibitor), the ROS scavenger N-acetylcysteine (NAC), and the pan-lipidation inhibitor 2-bromopalmitate (2BP) were tested. GSDMD, HIF-1α or STAT1 knockout (KO) BMDMs were evaluated. Plasma from PAH patients and healthy controls was analyzed for LDH activity as an indicator of cell lysis. Results: LDH release was significantly elevated in plasma from PAH patients as compared to controls, consistent with increased lytic cell death. In hypoxia-exposed BMDMs, inflammasome activation and pyroptotic cell death were increased under LPS+Ng treatment, as shown by elevated LDH release, Zombie Red staining, and IL-1β and HMGB1 secretion. Hypoxia also upregulated NLRP3 and IL-1β at mRNA levels. Pyroptosis was abolished by MCC950 in both normoxia and hypoxia, confirming that NLRP3 is required for this response. GSDMD KO BMDMs exhibited reduced pyroptosis, indicating that GSDMD is crucial for hypoxia-induced pyroptosis. Hypoxia-induced NLRP3 activation was dependent on HIF-1α, but not STAT1. Hypoxia alone and in combination with LPS increased ROS levels in macrophages, which was suppressed by NAC. NAC also blocked LDH release and pyroptosis markers, including caspase-1 and GSDMD activation. Additionally, 2BP reduced pyroptotic markers, implicating lipidation in the hypoxia-driven pyroptotic pathway. Conclusion: Our data demonstrate that hypoxia induces ROS production, enhances NLRP3 inflammasome expression and activation, thereby amplifying macrophage pyroptosis through ROS- and lipidation-dependent mechanisms. This newly described pathway provides insights into PAH pathogenesis and suggests hypoxia may be an important NLRP3 stimulus in vivo.
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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) | 0.001 | 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 | 0.001 | 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".