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: sepsis-induced lung injury, Maresin1, neutrophils, cellular
   heterogeneity

1. Introduction

   Sepsis is life-threatening multiple organ failure resulting from a
   dysregulated host response to infection, posing a serious worldwide
   health burden [[34]1]. Among the damaged organs, the lung is the most
   vulnerable organ during sepsis [[35]2] and sepsis-induced acute lung
   injury (ALI) is one of the most critical prognostic factors for
   mortality in sepsis patients [[36]3].

   Despite extensive research that was performed on sepsis-induced ALI,
   current treatment strategies are still dominated by supportive
   ventilation strategies. The pathophysiological mechanisms associated
   with the development and therapeutic targets of sepsis-induced ALI have
   so far not been definitively elucidated [[37]4].

   The inappropriate inflammatory response is an important pathological
   feature of septic lung injury. The resolution of inflammation was once
   thought to be a passive effect due to the reduction of pro-inflammatory
   mediators, while the discovery of the specialized pro-resolving
   mediators (SPMs) suggested that this process is a well-planned active
   and coordinated active process [[38]5,[39]6]. As a specific
   pro-inflammatory mediator derived from docosahexaenoic acid (DHA) in
   macrophages [[40]7], Maresin1 was demonstrated to have extensive
   regulatory effects on immune cells in inflammatory-related diseases
   including sepsis [[41]8]. We also performed some exploration on the
   role of Maresin1 during sepsis and our findings suggest that Maresin1
   can promote M2 macrophage polarization by promoting peroxisome
   proliferator-activated receptor-gamma activation [[42]9]. In addition,
   Maresin1 could reduce neutrophil accumulation and overcome
   Lipopolysaccharide-mediated inhibition of neutrophil apoptosis, etc.
   [[43]10,[44]11].

   Neutrophils have always been an important focus for exploring and
   solving the problem of septic lung injury. As an important component of
   the innate immune system, the overactivation of neutrophils is one of
   the important pathological mechanisms of septic lung injury [[45]12].
   Neutrophils can induce organ damage through a variety of pathways,
   including the release of inflammatory mediators such as cytokines and
   reactive oxygen species. The neutrophil infiltration was considered to
   be the rate-limiting step in the progression of ALI [[46]13].
   Neutrophils are heterogeneous [[47]14,[48]15,[49]16], especially in
   different environments such as homeostasis and bacterial infection,
   neutrophils show obvious heterogeneity [[50]17]. Exploring the
   heterogeneity of neutrophils in septic lung injury can help us discover
   whether targeting specific neutrophil subsets is a more effective
   treatment modality. However, current studies of neutrophils in septic
   lung injury have not distinguished their heterogeneity.

   Our previous studies have shown that Maresin1 has an effective
   protective effect on septic lung injury [[51]11,[52]18], which can
   significantly improve lung function and reduce the inflammatory
   response of lung tissue. However, it is unknown whether Maresin1 acts
   consistently on all subpopulations of neutrophils under inflammatory
   conditions. Single-cell RNA sequencing (scRNA-seq) is a powerful tool
   for exploring cellular heterogeneity [[53]19]. Therefore, by
   establishing the most commonly used sepsis model of cecal ligation and
   puncture (CLP), we used single-cell sequencing technology to reveal the
   regulatory mechanism of Maresin1 on neutrophils in septic lung injury.
   Our studies aimed to investigate the mechanism of Maresin1 regulation
   of neutrophils at the single-cell level in septic lung injury and to
   uncover the underlying cellular pathophysiologic mechanism.

2. Materials and Methods

2.1. Ethics Statement

   All animal experiments were approved by the Institutional Animal Care
   and Use Committee of Huazhong University of Science and Technology
   (IACUC Number: 2629).

2.2. Animals

   Twenty-one C57BL/6 male mice (six weeks old, 20–25 g), obtained from
   Beijing Weitong Lihua Experimental Animal Technical Co., Ltd., were
   randomly divided into 3 groups, including the Sham group, the CLP
   group, and the Maresin1 group. All mice were acclimated for one week
   before starting the experiment. The laboratory conditions were: a 12 h
   light–dark cycle, a humidity of 40–60%, and a temperature of 22–24 °C.
   All the mice were allowed free access to water and food.

   The construction of the CLP model is as we described in our previous
   studies [[54]20]. Briefly, we first anesthetized mice with 2% sodium
   pentobarbital at a dose of 80 mg/kg. We made a 1.5 cm incision along
   the midline of the mouse abdomen, took out the cecum, ligated the
   middle and perforated with a 20-gauge needle. After squeezing a little
   feces, the cecum was restored to the abdominal cavity, and the
   abdominal incision was sutured layer by layer. The mice in the Maresin1
   group were administered 1 ng of Maresin1 (Cayman Chemical, Ann Arbor,
   MI, USA; Cat.10878) in 100 μL of normal saline via the tail vein. Our
   previous studies [[55]11,[56]20] showed that 1ng of Maresin1 can exert
   an effective protective effect on mice with septic lung injury,
   therefore we choose 1 ng as the experimental dose. After 24 h, the
   abdominal aorta of the mice was cut and the mice were euthanized and
   the mouse specimens were collected for subsequent experiments.

2.3. The Lung Histological Examination

   The harvested lung tissue was fixed with 4% paraformaldehyde, embedded
   in paraffin, and cut into 4 μm sections. Then, the sections were
   deparaffinized and stained with hematoxylin and eosin (HE) (n = 7 per
   group). The sections were observed under the ordinary optical
   microscope and damage scores were calculated. The scoring method was
   based on the lung injury scoring criteria published by the American
   Thoracic Society [[57]21].

2.4. The Neutrophil Count in Bronchoalveolar Lavage Fluid (BALF)

   The right lung of the mouse was ligated and BALF was collected by
   lavage of the left lung with 1.5 mL of phosphate-buffered saline (PBS)
   (0.5 mL each). After BALF was centrifuged at 1000 rpm for 10 min, the
   cell pellet was resuspended with PBS and then stained with Giemsa stain
   (n = 7 per group). The number of neutrophils was counted under an
   ordinary optical microscope.

2.5. Immunofluorescence Assay

   The sections (4 μm-thick, n = 6 per group) were incubated with Ly6G
   antibody (Thermo Fisher Scientific, Waltham, MA, USA, 14-5931-81)
   (1:200) and the ICAM1 antibody (Abcam, Boston, MA, USA, ab222736)
   (1:100) followed by the fluorescently labeled secondary antibod (Wuhan
   baiqiandu Biotechnology Co., Ltd., Wuhan, China, 1:200, B1000804,
   B1000805). The positive cells were then observed under the Olympus
   fluorescence microscope. We observed five fields for each section and
   took the average value of the number of positive cells for statistics.

2.6. Single-Cell Sequenceing

   The three Sham group specimens, the three CLP group specimens, and the
   three Maresin1 group specimens were mixed, respectively, for
   single-cell RNA sequencing. Single-cell suspensions of lung tissue
   specimens were prepared according to OE Biotech Co., Ltd.’s advice
   [[58]22]. The cells were then sorted using Miltenyi mouse CD45 magnetic
   beads according to the manufacturer’s recommendations and sequenced at
   a 1:1 ratio. According to the manufacturer’s guidelines, the
   single-cell libraries were constructed by 10× Genomics Chromium Single
   Cell 3ʹ Reagent Kits v3.1. The Illumina Nova 6000 PE150 platform was
   used to sequence the libraries.

2.7. Single-Cell Data Preprocessing

   Cell Ranger v5.0.0 was used for the initial processing of the scRNA-seq
   data. We then performed the downstream bioinformatic analyses with
   Seurat v. 4.1.2 in the R statistical environment. The low-quality cells
   were removed if they expressed <2000 or >6000 genes/cell, UMI counts
   <1000 or mitochondrial UMI counts greater than 10% mitochondrial. The
   “Harmony” package was used to remove batch effects between samples
   [[59]23]. The cells after quality control were grouped with the
   “FindNeighbors” and “FindClusters” functions with a resolution of 1.
   The clusters were then visualized after “RunTSNE” or “RunUMAP”. The
   “FindMarkers” function in Seurat was used to identify DEGs. Only the
   genes that were expressed in more than 25% of the cells of a cluster
   and had an average log (fold change) value greater than 0.5 could be
   selected as DEGs. We annotated the cell type of each cluster by the
   expression of canonical markers found in DEGs. The cells expressing two
   or more canonical markers were considered double cells and also were
   eliminated.

   The final total was 11,025 immune cells, the Sham group was 2992, the
   CLP group was 4265, and the Maresin1 group was 3768.

2.8. Pathway Enrichment Analysis

   The gene ontology (GO) enrichment analysis [[60]24] and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) analysis [[61]25] were
   performed with the “clusterProfiler” R package [[62]26].

2.9. The Gene Regulatory Network Analysis

   The transcription factors were assessed by pyscenic [[63]27] analysis
   on all neutrophils and differentially expressed transcription factors
   were calculated using the Limma [[64]28] software package.

2.10. Statistical Analysis

   The validation data were obtained from the public functional genomics
   data repository of Gene Expression Omnibus ([65]GSE151974) [[66]29].
   The statistical analyses and graph generations were performed in R (v
   4.1.2) and GraphPad Prism (v 9.0).

3. Results

3.1. Maresin1 Attenuates the Extent of Sepsis-Induced Lung Damage

   As shown in [67]Figure 1A, compared with the Sham group, the mice in
   the CLP group had disordered lung tissue structure, thickened alveolar
   septa, accumulation of inflammatory cells in the alveoli, and the
   pulmonary septum, and bleeding and protein debris exudation in the
   alveolar cavity. The degree of lung injury of the mice in the Maresin1
   group was significantly reduced. The lung damage score was consistent
   with the pathological changes ([68]Figure 1B).

Figure 1.

   [69]Figure 1
   [70]Open in a new tab

   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.

3.2. Maresin1 Reduces Neutrophil Numbers in Bronchoalveolar Lavage

   As shown in [71]Figure 1C, the number of neutrophils in the BALF of the
   CLP group was significantly higher than that of the Sham group (p <
   0.01); compared with the CLP group, Maresin1 significantly decreased
   the number of neutrophils (p < 0.01) but was still higher than that of
   the Sham group (p < 0.01). The immunofluorescence results ([72]Figure
   1D) suggested that the recruitment and infiltration of the neutrophils
   in the lung tissue of the CLP group were significantly higher than in
   the Sham group, while Maresin1 could decrease the neutrophil
   infiltration. In order to observe the distribution of neutrophils more
   clearly, we performed fluorescent double staining of Ly6g and Icam1 and
   the results showed that neutrophils in septic lung injury were
   diffusely distributed ([73]Figure S1).

3.3. Maresin1 Alters Neutrophil Composition in Septic Lung Injury

   We use single-cell sequencing to reveal the immune microenvironment of
   lung tissue in septic lung injury. Based on the single-cell sequencing
   results, we first divided all immune cells in lung tissue into nine
   main types ([74]Figure 2A) according to the expression of classical
   markers ([75]Figure 2B), including: “Cycle cell”, “Alveolar
   Macrophages”, “Mono”, “Macrophages”, “DC cell”, “Neutrophil”, “B cell”,
   “Nk cell”, “T cell”. Among them, the Cycle cell subpopulation highly
   expresses Mki67 and Top2A [[76]30] and is a mixed population of immune
   cells in proliferating state. The top five DEGs of different clusters
   are shown in [77]Figure 2C. Furthermore, the whole DEGs of different
   immune cell clusters are listed in [78]Table S1. As shown in [79]Figure
   2A, neutrophils make up a large proportion of the immune cell
   population, therefore we further divided the neutrophils into four
   subpopulations ([80]Figure 2C) based on the marker genes ([81]Figure
   2D): Neutrophil0-Ccl6, Neutrophil1-Krt83, Neutrophil2-Csta2,
   Neutrophil3-Cxcl3. Notably, we found that the Neutrophil1-Krt83
   subpopulation highly expressed Krt83, which is recognized as the basic
   hair keratin gene [[82]31]. Therefore, we next used [83]GSE151974 for
   verification and we found that between the two subpopulations of
   neutrophils in [84]GSE151974, there was also a subpopulation that
   highly expressed Krt83 ([85]Figures S2A,B). Compared with normal mice,
   sepsis changed the proportion of all neutrophil subpopulations. The
   neutrophil fraction of the Maresin1 group, except for the
   Neutrophil3-Cxcl3 subgroup, was similar to that of the CLP group
   ([86]Figure 2E). Therefore, we selected the Neutrophil3-Cxcl3
   subpopulation for further analysis. The top 20 DEGs of the
   Neutrophil3-Cxcl3 subpopulation are shown in [87]Figure 3A. To further
   confirm the Neutrophil3-Cxcl3 subpopulation, we also performed
   validation by using [88]GSE151974. There are also neutrophils that
   express the Cxcl3 gene significantly in [89]GSE151974 ([90]Figure S2C).
   Functional analysis of the DEGs of the Neutrophil3-Cxcl3 subpopulation
   was subsequently performed. The GO analyses ([91]Figure 3B) suggested
   that the enrichment of DEGs was mainly involved in the Chemotaxis and
   recruitment of inflammatory cells, the release of cytokines, etc. The
   KEGG analyses ([92]Figure 3C) suggested that the enrichment of DEGs was
   mainly involved in the canonical pathways of neutrophils, including
   NF-kappa B signaling pathway, Cytokine–cytokine receptor interaction,
   and the Toll-like receptor signaling pathway. The functional analysis
   indicated that the Neutrophil3-Cxcl3 subpopulation was closely related
   to the excessive inflammatory response in septic lung injury.

Figure 2.

   [93]Figure 2
   [94]Open in a new tab

   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.

   [95]Figure 3
   [96]Open in a new tab

   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.

3.4. Maresin1 Inhibits the Expression of Key Genes in the Neutrophil-Cxcl3
Subpopulation

   Cxcl3, Ccl3, Ccl4, Icam1, and Il1rn are the main genes involved in the
   Go enrichment and KEGG pathway enrichment of the Neutrophil3-Cxcl3
   subpopulation. We extracted the Neutrophil3-Cxcl3 subpopulation from
   the total neutrophils for further analysis according to the source of
   the group. As shown in [97]Figure 4A, the expression of the Cxcl3,
   Ccl3, Ccl4, Icam1, and Il1rn of the Neutrophil3-Cxcl3 subpopulation was
   higher in the CLP group than in the Sham group, while Maresin1
   intervention decreased the expression of these genes.

Figure 4.

   [98]Figure 4
   [99]Open in a new tab

   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.

3.5. Maresin1 Represses Transcription Factors in Neutrophil-Cxcl3

   Transcription factors play a crucial role in cell differentiation and
   fate. Based on the results of pyscenic analysis and the previously
   published literature, we studied the transcription factors of
   neutrophils. Subpopulation analysis revealed that Irf5, Junb, Nfkb2,
   Relb, and Rfx2 transcription factors play important roles in the
   Neutrophil3-Cxcl3 subpopulation ([100]Figure 4B). Upon further analysis
   of the Neutrophil3-Cxcl3 subpopulation derived from different groups,
   we found that Maresin1 could significantly reduce the expression levels
   of these transcription factors ([101]Figure 4C).

4. Discussion

   Sepsis is a severe infectious complication characterized by a
   dysregulated immune response and subsequent multiple organ dysfunction
   [[102]32]. Although the exploration of the pathological mechanisms and
   therapeutic targets of septic lung injury has been ongoing in the past
   few decades, there is still no definite and effective treatment. Sepsis
   included the initial phase of hyper-inflammatory response followed by a
   phase of immunosuppression [[103]33]. When an acute inflammatory injury
   occurs, neutrophils can migrate and aggregate to the foci of the
   inflammatory reaction infection and play the role of capturing and
   phagocytizing microorganisms by releasing reactive oxygen species or
   through neutrophil extracellular nets (Nets). However, hyperactivated
   neutrophils are also an important factor in promoting the development
   and exacerbation of acute lung injury [[104]34]. Neutrophils are an
   important target for the treatment of acute lung injury in sepsis
   [[105]35].

   The discovery of SPMs provides new possibilities for the treatment of
   inflammatory-related diseases; however, the current regulation of
   immune cells by SPMs is not precise enough. As an important member of
   the SPMs family, Maresin1 has shown a positive prospect in the
   treatment of inflammatory-related diseases. On the one hand, Maresin1
   has a positive regulatory effect on a variety of inflammatory-related
   diseases. On the other hand, Maresin1 has regulatory effects on both
   innate and adaptive immune cells. However, the regulatory effect of
   Maresin1 on immune cells is still at the level of specific cell types
   and there is a lack of research on specific cell subclusters. Whether
   the regulatory effect of Maresin1 on immune cells is
   subpopulation-biased has not been demonstrated.

   Neutrophils are the most abundant leukocytes in mammals and play a key
   role in the pathogenesis of sepsis-induced lung injury [[106]36].
   However, there is considerable debate about the impact of
   therapy-induced neutrophil depletion in sepsis, as the impact on
   bacterial clearance and response to systemic inflammation is unclear
   [[107]37]. Another important explanation may be the obvious
   heterogeneity in the number ratio, functional characteristics,
   transcription factor expression, etc. of different subpopulations of
   neutrophils under inflammatory conditions [[108]17]. Therefore, in the
   diagnosis and treatment of septic lung injury, it is not enough to stay
   at the traditional cell type level. Positive interventions may not
   exert equal regulatory effects on one type of cell but rather have
   different regulatory potencies on different subpopulations of this type
   of cell.

   In the current study, we validated the role of Maresin1 in inhibiting
   neutrophil infiltration in septic lung injury and then focused on the
   subpopulation bias of Maresin1 regulation of neutrophils. We classified
   neutrophils in septic lung injury into four subpopulations based on
   distinct molecular signatures. We found that sepsis altered the
   proportion of different subsets of neutrophils in lung tissue, while
   Maresin1 intervention had a more pronounced effect on the proportion of
   the Neutrophil3-Cxcl3 subpopulation. We performed a DEGs analysis and
   functional analysis of the Neutrophil3-Cxcl3 subpopulation and found
   that the functions of DEGs in the Neutrophil3-Cxcl3 subpopulation
   mainly focus on cytokine receptor binding, regulation of cytokine
   production, regulation of cell–cell adhesion, and promotion of
   neutrophils, monocytes, and other immune cells chemotaxis and
   recruitment. The genes most involved in the functional enrichment
   analysis were Cxcl3, Ccl3, Ccl4, Icam1, and Il1rn. All of the Cxcl3,
   Ccl3, Ccl4, and Icam1 play an important role in the migration of
   neutrophils [[109]38,[110]39,[111]40,[112]41]. It is worth noting that
   the expression of Il1rn, as an anti-inflammatory gene [[113]42], was
   up-regulated in the CLP group, which may be caused by auto-protective
   regulation. Maresin1 can significantly reduce the expression of the
   Cxcl3, Ccl3, Ccl4, and Icam1 genes in the Neutrophil3-Cxcl3
   subpopulation, which may be an important mechanism by which Maresin1
   regulates neutrophil function, and also provides a possible
   intervention target for the treatment of septic lung injury.

   Relb, Irf5, and Junb are important transcription factors driving
   neutrophil effector responses, while Nfkb2, Rfx2, and Relb promote
   neutrophil survival [[114]43,[115]44,[116]45]. In addition to
   contributing to the expression and production of cytokines and
   chemokines, and various effector functions, Irf5, Junb, and Relb are
   also involved in regulating the transition from the blood into the
   tissue. Interestingly, the Neutrophil3-Cxcl3 subpopulation just
   specifically highly expressed Icam1 and a series of chemokines, such as
   Cxcl3, Ccl3, Ccl4, and so on. We deduced that the inflammatory
   conditions could lead to massive migration and infiltration of the
   Neutrophil3-Cxcl3 subpopulation into lung tissue. The recruitment of
   the Neutrophil3-Cxcl3 subpopulation will lead to the migration of other
   inflammatory cells to the lung tissue through the production of a
   series of cytokines and chemokines, forming an excessive inflammatory
   response, while Maresin1 can effectively inhibit this process.

   There are also certain limitations in our current study. Firstly,
   although we revealed that the regulatory effect of Maresin1 on
   neutrophils is subpopulation biased, our current study mainly explored
   the effect of Maresin1 on the transcriptional level of neutrophils. For
   the Neutrophil-Cxcl3 subpopulation, we validated the presence of
   neutrophils that highly express the Cxcl3 gene by using public datasets
   but we did not validate whether Cxcl3 is also highly expressed at the
   protein level. We have not yet explored the exact role of the
   Neutrophil-Cxcl3 subpopulation. Exploring the important role of the
   Neutrophil-Cxcl3 subpopulation requires new and substantial efforts,
   including siRNA infection, construction of Cxcl3 knockout animals, etc.
   Elucidating the role and mechanism of the Neutrophil-Cxcl3
   subpopulation is the focus of our next study. Secondly, our present
   study focused on the subpopulation on which Maresin1 has a major
   effect. While other interesting findings, such as the high expression
   of the keratin gene Krt83 in some neutrophils, were not explored. We
   will continue to explore this in depth in the future. In addition,
   another noteworthy problem is that Maresin1 is very unstable and needs
   to be stored at −80 °C. This problem seriously limits the application
   of Maresin1, which needs more exploration to overcome.

   In conclusion, we confirmed that the regulatory effect of Maresin1 on
   neutrophils during septic lung injury is subpopulation biased. Maresin1
   can effectively inhibit the infiltration of the Neutrophil3-Cxcl3
   subpopulation and inhibit the expression of key inflammatory genes and
   transcription factors in the Neutrophil3-Cxcl3 subpopulation. Our
   studies suggested that the exploration of sepsis lung injury should not
   only stop at the level of certain types of cells but also explore the
   heterogeneity of cells in depth, which is conducive to discovering the
   pathological mechanism of the disease and new treatment strategies.

Acknowledgments