Exploring the role of neutrophils in inflammatory pain hypersensitivity via single-cell transcriptome profiling

Abstract

Introduction: Myeloid CD11b⁺ cells are crucial mediators in post-operative and CFA-induced inflammation, but their role in pain, particularly the role of neutrophils, is still debated. This study employs single-cell RNA sequencing (scRNA-seq) to analyze CD11b⁺ cell composition in mice after surgery and CFA treatment and investigates the effects and mechanisms of Nicotinamide N-oxide (NAMO) on neutrophils and pain.

Methods: scRNA-seq was used to analyze the transcriptomes of CD11b⁺ cells in murine models of post-operative and CFA-induced inflammation. Using comprehensive bioinformatics techniques, we identified distinct cell subpopulations and characterized their gene expression profiles and functional attributes. Based on these analyses, NAMO was selected to intervene in neutrophil differentiation and maturation. The role of the CXCR2 target gene and NAMO in modulating post-operative and inflammatory pain was then evaluated, exploring potential mechanisms.

Results: scRNA-seq revealed a significant increase in neutrophils and a decrease in monocytes among CD11b⁺ cells following surgery and CFA treatment. Neutrophils comprised seven subpopulations at various differentiation stages from immature to mature. Given the high expression of CXCR2 in neutrophils, we used the CXCR2 inhibitor NAMO to suppress neutrophil differentiation and maturation, which subsequently alleviated post-operative and CFA-induced pain in mice. Proteomics analysis showed that NAMO treatment significantly reduced the expression of S100b and CaMKIIβ proteins in mouse neutrophils.

Discussion: Following surgery and CFA treatment, mature neutrophils were significantly elevated. The CXCR2 antagonist NAMO alleviated post-surgical and CFA-induced pain by inhibiting neutrophil differentiation and maturation. These findings offer novel approaches for pain prevention and treatment.

Keywords: CXCR2; NAMO; inflammation; neutrophil; pain.

Figure 1

scRNA-seq of CD11b⁺ cells from naïve, surgical, and CFA-treated mice. (A) Overview of the single-cell RNA sequencing methodology applied to CD11b⁺ cells. (B) Viability percentages of the nine samples evaluated. (C) UMAP visualization of CD11b⁺ cells isolated from naïve, surgical, and CFA-treated mice. (D) Comparative analysis of the composition of CD11b⁺ cells across the naïve, surgical, and CFA-treated groups. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc correction. ***P<0.001, ****P<0.0001 (relative to the naïve group). (E) Identification of the top five marker genes for various immune cell types: neutrophils, monocytes, T cells, B cells, NK cells, and basophils. (F) Functional annotation highlights of signature genes associated with the aforementioned immune cell subtypes.

Figure 2

scRNA-seq analysis of monocytes from naïve, surgical, and CFA-treated mice. (A) UMAP depiction of monocyte populations from naïve, surgical, and CFA-treated mice. (B) Distribution of monocytes across the M0-M3 subsets. (C) Expression profiles of the top five marker genes for the M0-M3 subsets. (D) Characterization of M0-M3 subsets on the basis of their marker gene expression. (E) Comparative analysis of M0-M3 subsets across different models via flow cytometry and single-cell RNA sequencing (percentages derived from panel b; mean ± SD; n = 3–6 mice from two independent experiments). (F) Monocle trajectory analysis of monocytes, illustrated by cluster identity (left) and pseudotime sequence (right) as coloring, with single-cell ordering on the basis of variable gene expression.

Figure 3

scRNA-seq analysis of neutrophils from naïve, surgical, and CFA-treated mice. (A) UMAP visualization of neutrophil subsets in naïve, surgical, and CFA-treated mice. (B) Composition analysis of neutrophils from naïve, surgical, and CFA-treated mice. (C) Monocle trajectory analysis of neutrophils, with color coding for cluster identity (left) and pseudotime sequence (right). Each dot represents a single cell, ordered by the expression of the most variable genes. (D) Heatmap displaying the expression levels of the top five genes defining the G0–G6 subsets. (E) Characterization of the G0-G6 subsets on the basis of marker gene expression. (F) Comparative analysis of neutrophil subsets in peripheral blood via flow cytometry and single-cell RNA sequencing across the three models.

Figure 4

Analysis of CXCL2-CXCR2 expression and functional scores in neutrophils. (A) Levels of CXCL2 and CXCR2 enrichment across various immune cell types in naïve, surgical, and CFA-treated mice. (B) Expression analysis of CXCL2 and CXCR2 specifically in neutrophils. (C) The functional scores of four-level particles of neutrophil in naïve, surgical, and CFA-treated mice. The four granule types are, in sequence: azurophil granules, specific granules, gelatinous granules and secretory vesicles. (D-I) Functional scores of neutrophil in naïve, surgical, and CFA-treated mice. Neutrophil degranulation (D), phagocytosis (E), chemotaxis (F), activation (G), apoptosis (H) and maturation (I). (J-O) Changes in neutrophil metabolism in naïve, surgical, and CFA-treated mice. (J-L) Changes in glycometabolism-related functions. Glucose metabolism (J), glycolysis (K) and pyruvate metabolism (L). (M-O) Scores of lipid metabolism related functions. Lipid metabolism (M), arachidonic acid metabolism (N) and triglyceride metabolism (O). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not statistically.

Figure 5

NAMO attenuates mechanical pain sensitization and delays neutrophil maturation. (A-D) The effects of NAMO on mechanical and thermal sensitization were evaluated following plantar incisions or CFA injections in NAMO-treated and control mice. The data are presented as the means ± SEMs (n = 8–15) and were analyzed via two-way repeated-measures ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control). (E, F) Giemsa staining was performed on Ly6G⁺ cells from peripheral blood. (E) Neutrophils transitioned from the mature state to the naïve state (magnification 100x). (F) Differential smears of neutrophils from NAMO-treated and control mice revealed alterations among mature, naïve, and intermediate neutrophil forms (magnification 40x). (G-J) Flow cytometry analysis was used to assess the impact of NAMO on neutrophils in blood and plantar tissues across two mouse models. The data are shown as the means ± SEMs and were analyzed via two-way repeated-measures ANOVA (*P < 0.05, n = 5–6 mice per group).

Figure 6

NAMO mitigates inflammation and neutrophil infiltration in plantar tissue. (A, B) Hematoxylin and eosin (H&E) staining was used to evaluate the inflammatory response in the plantar tissues of CFA-injected and incision-treated mice after NAMO administration. (C-E) Immunofluorescence staining was conducted to assess the infiltration of myeloid cells and neutrophils in the plantar tissues of CFA-injected and incision-treated mice following NAMO treatment.

Figure 7

Modulation of inflammation-associated cytokine and neutrophil protein expression by the NAMO. (A, B) Multiplex cytokine analyses were performed on plantar tissue following CFA injection and incisional treatment, both prior to and following NAMO administration (n = 8). The data were analyzed via two-way repeated-measures ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group). (C, E) Label-free proteomic analysis was conducted on Ly6G⁺ cells in the peripheral blood of CFA-treated mice. (C) A heatmap illustrates differential protein expression between the treatment groups. (D, E) Gene Ontology (GO) and gene set enrichment analysis (GSEA) were performed on differentially expressed proteins (DEPs) in Ly6G⁺ cells from CFA-treated mice.

Figure 8

Regulation of S100b and CaMKIIβ protein expression by NAMO. (A, B) Western blot analysis was used to measure the protein expression levels of S100b and CaMKIIβ in peripheral blood, plantar tissue, and Ly6G⁺ cells from mice that underwent surgery and CFA treatment, both before and after NAMO administration. (C, D) Immunohistochemical analysis was used to assess S100b protein expression in the plantar tissues of surgically treated and CFA-treated mice both before and after NAMO treatment. Cells with positive staining are indicated by a dark brown color (scale bar = 100 μm).