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. 2023 Jan 18:13:1062479.
doi: 10.3389/fimmu.2022.1062479. eCollection 2022.

Single-cell RNA-seq reveals cellular heterogeneity from deep fascia in patients with acute compartment syndrome

Tao Wang  1   2 , Yubin Long  1   2 , Lijie Ma  1   2 , Qi Dong  1   2 , Yiran Li  1   2 , Junfei Guo  1   2 , Lin Jin  1   2 , Luqin Di  1   2 , Yingze Zhang  1   2   3 , Ling Wang  1   2   4 , Zhiyong Hou  1   2   3
Affiliations

Single-cell RNA-seq reveals cellular heterogeneity from deep fascia in patients with acute compartment syndrome

Tao Wang et al. Front Immunol. .

Abstract

Introduction: High stress in the compartment surrounded by the deep fascia can cause acute compartment syndrome (ACS) that may result in necrosis of the limbs. The study aims to investigate the cellular heterogeneity of the deep fascia in ACS patients by single-cell RNA sequencing (scRNA-seq).

Methods: We collected deep fascia samples from patients with ACS (high-stress group, HG, n=3) and patients receiving thigh amputation due to osteosarcoma (normal-stress group, NG, n=3). We utilized ultrasound and scanning electron microscopy to observe the morphologic change of the deep fascia, used multiplex staining and multispectral imaging to explore immune cell infiltration, and applied scRNA-seq to investigate the cellular heterogeneity of the deep fascia and to identify differentially expressed genes.

Results: Notably, we identified GZMK+interferon-act CD4 central memory T cells as a specific high-stress compartment subcluster expressing interferon-related genes. Additionally, the changes in the proportions of inflammation-related subclusters, such as the increased proportion of M2 macrophages and decreased proportion of M1 macrophages, may play crucial roles in the balance of pro-inflammatory and anti-inflammatory in the development of ACS. Furthermore, we found that heat shock protein genes were highly expressed but metal ion-related genes (S100 family and metallothionein family) were down-regulated in various subpopulations under high stress.

Conclusions: We identified a high stress-specific subcluster and variations in immune cells and fibroblast subclusters, as well as their differentially expressed genes, in ACS patients. Our findings reveal the functions of the deep fascia in the pathophysiology of ACS, providing new approaches for its treatment and prevention.

Keywords: acute compartment syndrome; fibroblast; heat shock protein; immune cell; single cell RNA seq.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Clustering and classification of the cellular landscape of deep fascia tissue. (A) Overview of the experimental workflow. (B) tSNE visualization of cell cluster from deep fascia tissue. (C) Violin plots showing marker genes across cell clusters. (D) tSNE visualization of cell cluster from deep fascia tissue in HG and NG. (E) Bar plots showing the relative percentage of cell cluster for each sample as in (A). (F) Bar plots showing the relative percentage of cell cluster in HG and NG. scRNA-seq, single-cell RNA sequencing; HG, high stress group; NG, normal stress group.
Figure 2
Figure 2
Multiplex staining and multispectral imaging, ultrasound and Scanning Electron Microscope for deep fascia in two groups. (A) Multiplex staining and multispectral imaging showing fibroblasts and immune cell infiltration, including T cells and myeloid cells in HG compared with NG. *green (α-SMA): fibroblast; pink (CD14): myeloid cells; blue (CD3): T cell. (B) ultrasound in patients with in HG (right side) compared with the NG (left side) in ultrasound. (C) Scanning Electron Microscope in HG and NG. Left: parallel arrangement of collagen fiber bundles in NG; right: chaotic overall arrangement of collagen fiber bundles in HG. HG, high stress group; NG, normal stress group.
Figure 3
Figure 3
Clustering and classification of the T cell subclusters of deep fascia tissue. (A) tSNE visualization of T cell subclusters from deep fascia tissue. (B) Violin plots showing marker genes across T cell subclusters. (C) tSNE visualization of T cell subclusters from deep fascia tissue in HG and NG. (D) Bar plots showing the relative percentage of T cell subclusters for each sample. (E) Bar plots showing the relative percentage of T cell subclusters in HG and NG. HG, high stress group; NG, normal stress group; TCM, central memory T cell; Teff, effector T cell; Treg, regulator T cell; ILC=innate lymphoid cells; NKT=nature killer T cell.
Figure 4
Figure 4
Top 10 differentially expressed genes (DEGs) of T cell subclusters. TCM, central memory T cell; Teff, effector T cell; Treg, regulator T cell; ILC, innate lymphoid cells; NKT, nature killer T cell.
Figure 5
Figure 5
Clustering and classification of the myeloid cell subclusters from deep fascia tissue (A) tSNE visualization of myeloid cell subclusters from deep fascia tissue. (B) Violin plots showing marker genes across myeloid cell subclusters. (C) tSNE visualization of myeloid cell subclusters from deep fascia tissue in HG and NG. (D) Bar plots showing the relative percentage of myeloid cell subclusters for each sample. (E) Bar plots showing the relative percentage of myeloid cell subclusters in HG and NG. HG, high stress group; NG, normal stress group; Mac, macrophage; DCs, dendritic cells; Mon, monocyte.
Figure 6
Figure 6
Top 10 differentially expressed genes (DEGs) of myeloid cell subclusters in HG and NG. (A) DEGs of macrophage (Mac) subclusters in HG and NG. (B) DEGs of dendritic cells (DCs) subclusters in HG and NG. (C) DEGs of monocyte (Mon) subclusters in HG and NG. HG, high stress group; NG, normal stress group.
Figure 7
Figure 7
Clustering and classification of the fibroblasts (Fib) subclusters of deep fascia tissue (A) tSNE visualization of fibroblasts subclusters from deep fascia tissue. (B) Violin plots showing marker genes across fibroblasts subclusters. (C) tSNE visualization of fibroblasts subclusters from deep fascia tissue in HG and NG. (D) Bar plots showing the relative percentage of fibroblasts subclusters for each sample. (E) Bar plots showing the relative percentage of fibroblasts subclusters in HG and NG. HG, high stress group; NG, normal stress group.
Figure 8
Figure 8
Top 10 differentially expressed genes (DEGs) of fibroblasts (Fib) subclusters in HG and NG. HG, high stress group; NG, normal stress group.
Figure 9
Figure 9
Crosstalk among T cell, fibroblasts and myeloid cell. (A) Heatmap presents the key cellular interaction between T cell and myeloid cell. (B) Heatmap presents the key cellular interaction between T cell and fibroblasts. (C) Heatmap presents the key cellular interaction between fibroblasts and myeloid cell. (D) Dot plot shows the significant ligand-receptor pairs between T cell subsubsets and myeloid cell subsubsets. (E) Dot plot shows the significant ligand-receptor pairs between T cell subsubsets and fibroblasts subsubsets. (F) Dot plot shows the significant ligand-receptor pairs between myeloid cell subsubsets and fibroblasts subsubsets.
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