What question did this study set out to answer?

The research aims to understand the mechanisms driving pro-inflammatory macrophage activation in acute lung injury (ALI).

March 25, 2026Open Access

The CXCR2–XBP1–NF-κB Axis Drives Inflammatory Amplification and Tissue Damage in Acute Lung Injury

Key Points

The research aims to understand the mechanisms driving pro-inflammatory macrophage activation in acute lung injury (ALI).
Analyzed single-cell RNA sequencing of lung tissues from LPS-induced ALI mice to assess immune cell types.
Conducted in vitro experiments with bone marrow-derived macrophages using Cxcr2 overexpression and Xbp1 knockdown.
Utilized luciferase reporter assays and immunoblotting to evaluate NF-κB activation.
Implemented AAV-mediated genetic alterations to manipulate Cxcr2 and Xbp1 in mice before LPS exposure.
Evaluated outcomes through histopathology, cytokine measurement, and molecular profiling.
Identified a population of Cxcr2-expressing macrophages that expanded in ALI and showed increased inflammation.
Cxcr2 overexpression enhanced M1 macrophage polarization via XBP1-induced ER stress, driving ongoing NF-κB activation.
CXCR2 inhibition reduced lung injury and M1 macrophage levels in vivo.
AAV-driven Cxcr2 overexpression worsened inflammation, while Xbp1 knockdown improved pathological features.

Abstract

Acute lung injury (ALI) is a critical pulmonary disorder marked by severe inflammation, alveolar-capillary barrier disruption, and respiratory failure. Macrophage polarization is central to its pathogenesis, yet the underlying regulatory mechanisms remain incompletely understood. This study aimed to identify and functionally characterize key drivers of pro-inflammatory macrophage activation in ALI. We utilized an integrative multi-platform approach. First, single-cell RNA sequencing of lung tissues from LPS-induced ALI mice was analyzed to map immune cell heterogeneity. Bone marrow-derived macrophages were then used for in vitro mechanistic studies, including Cxcr2 overexpression and Xbp1 knockdown, followed by transcriptomic and signaling analyses. NF-κB activation was assessed through luciferase reporter assays and phospho-specific immunoblotting. Finally, adeno-associated virus (AAV)-mediated genetic interventions—overexpressing Cxcr2 or silencing Xbp1—were delivered intratracheally in mice prior to LPS challenge, with outcomes evaluated by histopathology, cytokine measurement, and molecular profiling. Our analysis revealed a dominant population of Cxcr2-expressing macrophages that expands during ALI and exhibits a hyper-inflammatory signature. Cxcr2 overexpression amplified M1 polarization via XBP1-mediated endoplasmic reticulum stress, leading to sustained NF-κB activation and cytokine hypersecretion. Pharmacological CXCR2 inhibition attenuated lung injury and reduced M1 macrophages in vivo. Conversely, AAV-driven Cxcr2 overexpression exacerbated inflammation, whereas Xbp1 knockdown significantly mitigated pathological features. These results establish the CXCR2–XBP1 signaling axis as a critical regulator of pathological macrophage polarization in ALI, acting through ER stress and NF-κB hyperactivation. Our study highlights CXCR2 and XBP1 as potential therapeutic targets for modulating macrophage-driven immunopathology.

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