C94-08 Single-Cell Analysis Reveals Transcriptional Changes Linked to Impaired Alveolar Macrophage Efferocytosis Following Acute Lung Injury Induced by Ozone and Endotoxin

Abstract Rationale Ozone exposure has been linked to the development of acute respiratory distress syndrome (ARDS) in patients with sepsis. In a murine model, we demonstrated that inhalation of ozone prior to intravenous administration of lipopolysaccharide (LPS) to model sepsis, induces neutrophilic inflammation and acute lung injury (ALI). Alveolar macrophages (AMs) have a critical role in resolving inflammation by phagocytosing apoptotic neutrophils (PMNs)—a process known as efferocytosis—which depends on surface receptors such as MerTK. We have demonstrated that ozone exposure reduces AM efferocytosis of PMNs, coinciding with MerTK receptor shedding. To elucidate transcriptional changes in myeloid subpopulations that also contribute to impaired efferocytosis, we performed single-cell RNA sequencing. Methods Male C57Bl/6J mice were exposed to 0.8 ppm ozone or filtered air for 3 hours in a whole-body chamber. After 24 hours, mice received LPS (3 mg/kg, i.v.) or PBS as a control. Bronchoalveolar lavage cells were collected 24 hours post-treatment from pooled samples (2-3 mice/group) across 3 replicate experiments. Cells were processed for single-cell RNA sequencing using the 10x Genomics Chromium platform and sequenced with Illumina technology. Cell clusters were identified using a nearest-neighbor approach and visualized via Uniform Manifold Approximation and Projection (UMAP). Results Among 22 identified cell clusters, 4 were classified as PMNs and 12 as AMs based on characteristic markers (Mertk, Mrc1, Itgax, Pparg, Adgre1, Itgam, CD14, S100a9, Sell). Mice exposed to ozone + LPS showed a marked increase in PMN frequency compared to air + PBS, air + LPS, or ozone + PBS groups (69.5% vs. 3.4%, 5.4%, and 4.4%, respectively). Re-clustering of PMNs revealed 10 subpopulations, with cluster 3—characterized by elevated Cd274 expression (encoding PD-L1, an anti-apoptotic checkpoint receptor)—being enriched in the ozone + LPS group. Of the 12 AM clusters, 2 clusters expressed relatively high levels of Mertk. These AM subsets were less abundant in ozone + LPS exposed mice. Pseudo-bulk analysis revealed reduced Mertk transcription across both high and low Mertk-expressing AM subsets in this group. Conclusion These data demonstrate that after exposure to ozone + LPS, there is diversity in PMN and AM subset expression of genes associated with efferocytosis. Moreover, overall Mertk transcription was reduced in these subsets, suggesting that there is no genetic compensation for the shedding of AM MerTK receptors. Additionally, we identified a subgroup of PMNs that may evade apoptosis, and thus bypass efferocytosis leading to prolonged neutrophilia in the lung and exacerbation of ALI. This abstract is funded by: NIH