Single-Cell Sequencing Reveals the Regulatory Role of Maresin1 on Neutrophils during Septic Lung Injury

Abstract

Acute lung injury (ALI) is the most common type of organ injury in sepsis, with high morbidity and mortality. Sepsis is characterized by an inappropriate inflammatory response while neutrophils exert an important role in the excessive inflammatory response. The discovery of specialized pro-resolving mediators (SPMs) provides a new direction for the treatment of a series of inflammatory-related diseases including sepsis. Among them, the regulation of Maresin1 on immune cells was widely demonstrated. However, current research on the regulatory effects of Maresin1 on immune cells has remained at the level of certain cell types. Under inflammatory conditions, the immune environment is complex and immune cells exhibit obvious heterogeneity. Neutrophils play a key role in the occurrence and development of septic lung injury. Whether there is a subpopulation bias in the regulation of neutrophils by Maresin1 has not been elucidated. Therefore, with the well-established cecal ligation and puncture (CLP) model and single-cell sequencing technology, our study reveals for the first time the regulatory mechanism of Maresin1 on neutrophils at the single-cell level. Our study suggested that Maresin1 can significantly reduce neutrophil infiltration in septic lung injury and that this regulatory effect is more concentrated in the Neutrophil-Cxcl3 subpopulation. Maresin1 can significantly reduce the infiltration of the Neutrophil-Cxcl3 subpopulation and inhibit the expression of related inflammatory genes and key transcription factors in the Neutrophil-Cxcl3 subpopulation. Our study provided new possibilities for specific modulation of neutrophil function in septic lung injury.

Keywords: Maresin1; cellular heterogeneity; neutrophils; sepsis-induced lung injury.

Figure 1

Maresin1 attenuates lung injury and neutrophil infiltration in septic lung injury. (A): The representative lung tissue HE staining results of different groups of mice (scale: 50 μm, magnification 200×). (B): The number of neutrophils in the BALF was counted by using the Wright–Giemsa method. (C): The lung injury scores of all groups. (D): The relative proportions of neutrophils in different groups of BALF counted by the Wright–Giemsa method. the results of the neutrophil count in BALF. (E): The statistical results of neutrophil immunofluorescence staining corresponding to (F). (F): The immunofluorescence staining results of the neutrophils in the lung tissues e (scale: 50 μm, magnification 200×). Data were presented as means ± SEM. ** p < 0.01 versus the Sham group; ## p < 0.01 versus the CLP group.

Figure 2

Single-cell sequencing revealed the cellular component of the neutrophil populations. (A): The tsne plot of all immune cells (Cd45+), showing a total of 9 distinct immune cell types that were identified. The different cell types are colored as indicated on the label. (B): The dot plot of marker genes for different types of immune cells. (C): The top5 DEGs for different immune cell clusters. (D): The umap plot of the cellular composition of neutrophils in lung tissue. A total of four clusters of neutrophils were identified. (E): The dot plot for the principal marker genes of different neutrophil subpopulations. (F): The fraction of different neutrophil subpopulations.

Figure 3

The DEGs and functional analysis of Neutrophil-cxcl3 subpopulation. (A): The heat map of the expression levels of the top20 DEGs of the Neutrophil-cxcl3 subpopulation. (B): The Go enrichment analysis of the DEGs of the Neutrophil-cxcl3 subpopulation. (C): The KEGG enrichment analysis of the DEGs of Neutrophil-cxcl3 subpopulation.

Figure 4

Maresin1 regulates gene expression and transcription factor expression of the neutrophil-Cxcl3 subset. (A): The heat map of the average expression levels of related genes for different groups. (B): The heat map of the regulon activity scores of TF motifs estimated per cell by pSCENIC. Shown are five differential activated motifs in the Neutrophil-cxcl3 subpopulation. (C): Maresin1 suppresses the expression levels of key transcription factors of the Neutrophil-cxcl3 subpopulation.