25-Hydroxycholesterol exacerbates vascular leak during acute lung injury

JCI Insight · 15 authors, 6 centres

AI SUMMARY

FIDELITY 90%

POPULATIONMice (Ch25h+/+ and Ch25h–/–, C57BL/6 background) exposed to high-dose LPS aerosol or Klebsiella pneumoniae lung infection; human ARDS patients from a phase II trial of omega-3 fatty acids (n=30 for AM CH25H, n=81 for BALF 25HC).

INTERVENTIONGenetic deletion of Ch25h (Ch25h–/– mice); exogenous 25HC administration (i.p. 50 mg/kg); ER stress relief with 4-phenylbutyric acid (PBA, 100 mg/kg i.p.); bone marrow chimeras to dissect hematopoietic vs. non-hematopoietic CH25H contribution.

COMPARISONCh25h+/+ (wild-type) vs. Ch25h–/– mice; vehicle vs. 25HC treatment; PBS vs. PBA treatment; WT>WT vs. KO>WT vs. WT>KO vs. KO>KO bone marrow chimeras.

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TL;DR

This study demonstrates that 25-hydroxycholesterol (25HC), an oxysterol induced during lung inflammation, exacerbates vascular leak in acute lung injury by promoting endothelial barrier dysfunction through LXR-independent ER stress. In mouse models of severe lung injury, genetic deletion of Ch25h (the enzyme that produces 25HC) markedly reduced microvascular leak, cytokine levels, and markers of endothelial injury. In patients with ARDS, higher alveolar macrophage CH25H expression and BALF 25HC levels correlated with increased alveolar protein, von Willebrand factor, and clinical disease severity, suggesting this pathway may be a therapeutic target.

Full summary

3,656 CHARS

**Background:** 25-hydroxycholesterol (25HC) is an oxysterol produced by cholesterol-25-hydroxylase (CH25H) that has been implicated in immune regulation. While prior work showed that 25HC promotes resolution of mild lung inflammation via LXR-dependent efferocytosis, its role in severe lung injury was unknown. Ch25h is most highly expressed in the lung, particularly in alveolar macrophages and, as shown here, in pulmonary endothelial cells. This study investigates whether 25HC contributes to acute lung injury and vascular leak in models of severe inflammation and in human ARDS.

**Methods:** The authors used a high-dose LPS aerosol model (3 mg/mL, 30 min) and Klebsiella pneumoniae lung infection in Ch25h+/+ and Ch25h–/– mice. They assessed bronchoalveolar lavage fluid (BALF) protein, albumin, IgM, cytokines, and cellular infiltration. Bone marrow chimeras were generated to determine the cellular source of pathogenic 25HC. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with 25HC ± LPS or heat-killed S. aureus, and transendothelial electrical resistance (TER) and VE-cadherin localization were measured. ER stress markers were quantified by qPCR. In human ARDS patients (n=30 for AM CH25H; n=81 for BALF 25HC), associations with alveolar protein, von Willebrand factor, PaO2/FiO2 ratio, and Lung Injury Score were assessed by linear regression adjusted for age, sex, APACHE II, and treatment group.

**Key Results:** Ch25h–/– mice exhibited marked and sustained reductions in BALF protein, albumin, and IgM after high-dose LPS (e.g., BALF protein ~2–3 mg/mL in Ch25h+/+ vs. ~1 mg/mL in Ch25h–/– at 48–72 h, P<0.01–0.001). BALF cytokines (IL-6, IL-12p40, G-CSF, CCL5, others) were broadly reduced in Ch25h–/– mice at 24–72 h. Similar protection from vascular leak was seen after K. pneumoniae infection. LXR-null mice showed no protection, indicating LXR independence. Bone marrow chimeras revealed that non-hematopoietic cell Ch25h was the primary driver of vascular leak; endothelial cells (CD45–EpCAM–CD31+) sorted from LPS-exposed lungs showed robust Ch25h induction. Exogenous 25HC (i.p.) induced lung Vcam1 in naive mice. In vitro, 25HC (20 μM) decreased TER in HPAECs and exacerbated LPS- and HKSA-induced barrier disruption. 25HC caused VE-cadherin disorganization and increased FITC-avidin permeability. Ch25h–/– lungs had markedly lower Chop (Ddit3) expression after LPS (P<0.001). Systemic 25HC induced Chop, BiP, and Atf4 in naive lungs. BALF from Ch25h+/+ but not Ch25h–/– mice induced Chop and Atf4 in cultured endothelial cells. Treatment with the ER stress reliever PBA (100 mg/kg) reduced BALF IgM and albumin in Ch25h+/+ but not Ch25h–/– mice, equalizing leak between genotypes. In human ARDS, AM CH25H expression correlated with BIP, ATF6, and XBP1 (all P<0.05). Higher AM CH25H and BALF 25HC were associated with increased BALF total protein and von Willebrand factor (P<0.05 for all, adjusted). Higher CH25H and 25HC were also associated with worse PaO2/FiO2 ratio (P<0.05) and higher Lung Injury Score (P<0.05).

**Clinical Implications:** This study identifies 25HC as a dual-role mediator in lung inflammation—pro-resolving in mild injury but pro-injury in severe injury—acting through endothelial ER stress and barrier disruption. The findings suggest that CH25H/25HC may be a biomarker of ARDS severity and a potential therapeutic target, though the context-dependent effects (LXR-dependent efferocytosis vs. LXR-independent endothelial injury) indicate that pharmacologic targeting will be challenging. The human data, while correlative, support the translational relevance of this pathway.

PICO

PPOPULATION

Mice (Ch25h+/+ and Ch25h–/–, C57BL/6 background) exposed to high-dose LPS aerosol or Klebsiella pneumoniae lung infection; human ARDS patients from a phase II trial of omega-3 fatty acids (n=30 for AM CH25H, n=81 for BALF 25HC).

IINTERVENTION

Genetic deletion of Ch25h (Ch25h–/– mice); exogenous 25HC administration (i.p. 50 mg/kg); ER stress relief with 4-phenylbutyric acid (PBA, 100 mg/kg i.p.); bone marrow chimeras to dissect hematopoietic vs. non-hematopoietic CH25H contribution.

OOUTCOME

BALF protein, albumin, IgM (microvascular leak); BALF cytokines; serum angiopoietin-2; lung VE-cadherin; transendothelial electrical resistance (TER); ER stress markers (Chop, BiP, Atf4); in humans: BALF total protein, von Willebrand factor, PaO2/FiO2 ratio, Lung Injury Score.

STUDY TYPE

other

SPECIALTY

pulmonology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

90% · A

ORIGINAL PAPER

PMC10132150

25-Hydroxycholesterol exacerbates vascular leak during acute lung injury

JCI Insight

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ABSTRACT

Abstract Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined. Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes liver X receptor–dependent (LXR-dependent) clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h –/– mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains proinflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h , and non-hematopoietic Ch25h deletion is sufficient to confer lung protection. In patients with acute respiratory distress syndrome, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting on different cell types in the lung communicates distinct, temporal LXR-independent and -dependent signals to regulate inflammatory homeostasis. Keywords: Inflammation, Pulmonology Keywords: Cell stress, Innate immunity, Mouse models

CITATION EXPORT

Jennifer H Madenspacher, Eric D Morrell, Jeffrey G McDonald et al.. 25-Hydroxycholesterol exacerbates vascular leak during acute lung injury. JCI Insight. (2023). https://doi.org/10.1172/jci.insight.155448

AUTHORS

15 · 6 CENTRES

Jennifer H Madenspacher, Eric D Morrell, Jeffrey G McDonald, Bonne M Thompson, Yue Li, Konstantin G Birukov, Anna A Birukova, Renee D Stapleton, Aidin Alejo, Peer W Karmaus, Julie M Meacham, Prashant Rai, Carmen Mikacenic, Mark M Wurfel, Michael B Fessler

1Immunity, Inflammation, and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA.
2Section of Pulmonary, Critical Care, and Sleep Medicine, Harborview Medical Center, Seattle, Washington, USA.
3Center for Human Nutrition and
4Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
5Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
6Department of Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont, USA.