Single-cell RNA sequencing analysis reveals the relationship of bone marrow and osteopenia in STZ-induced type 1 diabetic mice

Abstract

Introduction: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Broad knowledge about the genetics, epidemiology and clinical management of T1D has been achieved, but understandings about the cell varieties in the bone marrow during T1D remain limited.

Objectives: We aimed to present a profile of the bone marrow cells and reveal the relationship of bone marrow and osteopenia in streptozotocin (STZ)-induced T1D mice.

Methods: The whole bone marrow cells from the femurs and tibias of healthy (group C) and STZ-induced T1D mice (group D) were collected for single-cell RNA sequencing analysis. Single-cell flow cytometry and immunohistochemistry were performed to confirm the proportional changes among bone marrow neutrophils (BM-neutrophils) (Cxcr2⁺, Ly6g⁺) and B lymphocytes (Cd19⁺). X-ray and micro-CT were performed to detect bone mineral density. The correlation between the ratio of BM-neutrophils/B lymphocytes and osteopenia in STZ-induced T1D mice was analyzed by nonparametric Spearman correlation analysis.

Results: The bone marrow cells in groups C and D were divided into 12 clusters, and 249 differentially expressed genes were found. The diversity of CD45⁺ immune cells between groups C and D were greatly affected: the proportion of BM-neutrophils showed a significant increase while the proportion of B lymphocytes in group D showed a significant decrease. X-ray and micro-CT analyses confirmed that osteopenia occurred in group D mice. In addition, the results of single-cell flow cytometry and correlation analysis showed that the ratio of BM-neutrophils/B lymphocytes negatively correlated with osteopenia in STZ-induced T1D mice.

Conclusion: A single-cell RNA sequencing analysis revealed the profile and heterogeneity of bone marrow immune cells in STZ-induced T1D mice for the first time. The ratio of BM-neutrophils/B lymphocytes negatively correlated with osteopenia in STZ-induced T1D mice, which may enhance understanding for treating T1D and preventing T1D-induced osteopenia.

Keywords: Bone marrow; Lymphocyte; Neutrophil; Osteopenia; Single-cell RNA sequencing; Type 1 diabetes.

Graphical abstract

Fig. 1

A single-cell RNA sequencing revealed the atlas of bone marrow cells in healthy and STZ-induced T1D mice. (A) Study overview. (B-D) Twelve bone marrow cells clusters. UMAP of unbiased clustering of all bone marrow cells in groups C and D (B). UMAP of unbiased clustering and cell annotation of bone marrow cells in group C (C). UMAP of unbiased clustering and cell annotation of bone marrow cells in group D (D). (E) The proportions of each cell in groups C and D. (F) The specific proportion of CD45⁺ cells in groups C and D. (G) Key cell type marker genes of 12 cell clusters, the redder the color, the higher the expression. (H) Cluster signature genes highlighted on left. Expression of top differentially expressed genes (rows) across the cells (columns), the warmer the color, the higher the expression.

Fig. 2

The characteristics of BM-neutrophils between groups C and D. (A) UMAP of BM-neutrophil cluster among all cells. (B-C) Six BM-neutrophil subsets. UMAP of unbiased clustering and cell annotation of BM-neutrophil subsets in groups C and D (B). UMAP of unbiased clustering of BM-neutrophil subsets in groups C and D (C). (D) The proportion of BM-neutrophil subsets in groups C and D. (E) BM-neutrophils were labeled by immunohistochemistry of the sections of femur in groups C and D (CXCR2; Scale bar, 40 μm or 20 μm) (F) Key marker genes of BM-neutrophil subsets. UMAP of key marker genes of BM-neutrophil subsets, along with the corresponding distribution of expression levels among 12 clusters and 6 BM-neutrophil subsets respectively. Key marker genes included Asprv1, Ltf, Clec4d, Ube2c, Pcna and Actb.

Fig. 3

Differential trajectory of BM-neutrophil subsets and enrichment of DEGs. (A-D) Differential directions of 6 BM-neutrophil subsets. Heatmap presenting relative expressions of markers of BM-neutrophils along inferred trajectories in 6 BM-neutrophil subsets. The red and blue branches correspond to the two differential directions (A). Monocle pseudotime trajectory expression pattern of 6 BM-neutrophil subsets (B). Pseudotime trajectories of the BM-neutrophil subsets (C-D). (E) Latent time and RNA velocity of 6 BM-neutrophil subsets. Latent time and RNA velocity showed the internal clock of BM-neutrophil subsets. Different colors of latent time represent different differentiation times, the colder the color, the earlier, the warmer the color, the later; The arrows of RNA velocity represent the different directions of differentiation. (F) Signature genes of BM-neutrophil subsets. Subset signature genes highlighted on left. Expression of signature genes (rows) across the cells (columns), the warmer the color, the higher the expression. (G) GO analysis of BM-neutrophil_1. Bubble diagram of upregulated DEGs of BM-neutrophil_1 enriched in GO analysis. (H) KEGG analysis of BM-neutrophil_1. Bubble diagram of upregulated DEGs of BM-neutrophil_1 enriched in KEGG analysis. (I-J) The top 5 DEGs of BM-neutrophils. The top 5 upregulated DEGs between groups C and D (I). The top 5 downregulated DEGs between groups C and D (J).

Fig. 4

The characteristics of B lymphocytes between groups C and D. (A) UMAP of the cluster of B lymphocytes among all cells. (B-C) Two B lymphocyte subsets. UMAP of unbiased clustering and cell annotation of the subsets of B lymphocytes in groups C and D (B). UMAP of unbiased clustering of the subsets of B lymphocytes in groups C and D (C). (D) The proportion of the subsets of B lymphocytes in groups C and D. (E) B lymphocytes were labeled by immunohistochemistry of the sections of femur in groups C and D (CD79a; Scale bar, 40 μm or 20 μm). (F) Key marker genes of B lymphocyte subsets. UMAP of key marker genes of the subsets of B lymphocytes, along with the corresponding distribution of expression levels among 12 clusters. Key marker genes included Bank1, CD83 and Ighd. (G) Latent time and RNA velocity of 3B lymphocyte subsets. Latent time and RNA velocity showed the internal clock of B lymphocyte subsets. Different colors of latent time represent different differentiation times, the colder the color, the earlier, the warmer the color, the later; The arrows of RNA velocity represent the different directions of differentiation. (H) GO analysis of Naïve_B. Bubble diagram of upregulated DEGs of Naïve_B enriched in GO analysis. (I) KEGG analysis of Naïve_B. Bubble diagram of upregulated DEGs of Naïve_B enriched in KEGG analysis. (J-K) The top 5 DEGs of B lymphocytes. The top 5 upregulated DEGs between groups C and D (J). The top 5 downregulated DEGs between groups C and D (K).

Fig. 5

The characteristics of monocytes, T lymphocytes and DCs between groups C and D. (A-B) Five monocyte subsets. UMAP of unbiased clustering and cell annotation of the subsets of monocytes in groups C and D (A). UMAP of unbiased clustering of the subsets of monocytes in groups C and D (B). (C) The proportion of the subsets of monocytes in groups C and D. (D) Signature genes of monocyte subsets. Subset signature genes highlighted on left. Expression of signature genes (rows) across the cells (columns), the warmer the color, the higher the expression. (E-F) Four T lymphocyte subsets. UMAP of unbiased clustering and cell annotation of the subsets of T lymphocytes in groups C and D (E). UMAP of unbiased clustering of the subsets of T lymphocytes in groups C and D (F). (G) The proportion of the subsets of T lymphocytes in groups C and D. (H) Signature genes of T lymphocyte subsets. Subset signature genes highlighted on left. Expression of signature genes (rows) across the cells (columns), the warmer the color, the higher the expression. (I-J) Four dendritic cell subsets. UMAP of unbiased clustering and cell annotation of the subsets of DCs in groups C and D (I). UMAP of unbiased clustering of the subsets of DCs in groups C and D (J). (K) The proportion of the subsets of DCs in groups C and D. (L) Signature genes of dendritic cell subsets. Subset signature genes highlighted on left. Expression of signature genes (rows) across the cells (columns), the warmer the color, the higher the expression.

Fig. 6

Osteopenia occurred in STZ-induced T1D mice. (A) X-ray imaging of left legs in groups C and D. It can be seen intuitively that the BMD of group C was higher than that of Group D. Window level was set from 2058 to 7200. (B) Micro-CT imaging of the same part of femurs in groups C and D. It can be seen intuitively that the bone cortex of group C was thicker than that of group D. (C) Micro-CT analysis. The results showed BMD (***p < 0.001), BV/TV (***p < 0.001), Tb.Th (***p < 0.001), Tb.N (p = 0.098) and Tb.Sp (p = 0.085).

Fig. 7

Identification of STZ-induced T1D mice and the ratio of immune cells between groups C and D. (A) The weight and blood sugar between group C and D. The weight and blood sugar of STZ-induced T1D mice were detected at ages of 22 W and 28 W (**p < 0.01, ***p < 0.001). (B) The ratio of different immune cells. The ratio of BM-neutrophils/T lymphocytes, BM-neutrophils/B lymphocytes, BM-neutrophils/DCs, BM-neutrophils/monocytes, monocytes/T lymphocytes, monocytes/B lymphocytes, monocytes/DCs, BM-neutrophil_1/Naïve_B in groups C and D. (C) The RD/RC among different immune cells. The RD/RC of BM-neutrophils/T lymphocytes, BM-neutrophils/B lymphocytes, BM-neutrophils/DCs, BM-neutrophils/monocytes, monocytes/T lymphocytes, monocytes/B lymphocytes, monocytes/DCs, BM-neutrophil_1/Naïve_B.

Fig. 8

The correlation between immune cells and osteopenia. (A) Flow cytometry of BM-neutrophils and B lymphocytes from bone marrow. The proportion of BM-neutrophils increased notably in group D and the proportion of B lymphocytes decreased notably in group D. (B) The ratios of BM-neutrophils/B lymphocytes from the results of flow cytometry. The ratio of BM-neutrophils/B lymphocytes was notably higher in group D (p = 0.040). (C) The correlation analysis between the ratio of BM-neutrophils/B lymphocytes and the ratio of BV/TV. There was a negative correlation between the ratio of BV/TV and the ratio of BM-neutrophils/B lymphocytes. (r = -0.7619, p = 0.0368). (*p < 0.05).