Lepr db/db mice show early-onset hyposensitivity and hyperglycemia

Von Frey testing of WT and Leprdb/db mice showed significant hyposensitivity in Leprdb/db mice ranging from 11 to 21 weeks of age (Fig. 1a). In addition, blood glucose of mice at 12 weeks of age confirmed a significant hyperglycemia in Leprdb/db mice by 12 weeks of age (Fig. 1b).

Fig. 1

Model validation, analysis pipeline, and identification of hindpaw immune cell heterogeneity: a von Frey and b blood glucose levels were used to verify the neuropathy and metabolic phenotype of Leprdb/db mice, respectively (n = 5). c Overview of the single-cell RNA-seq workflow. d Gating strategy for the isolation of single cells from hindpaws by flow cytometry depicting the gating system used to isolate CD45+ cells, which included a gate based on forward (FSC) and side (SSC) scatter, followed by a singlet gate and a CD45+ gate. In addition to these gates, which were used for sample collection, a CD19+ gate was used to check for excessive B-cell numbers as a proxy measure of bone marrow contamination. e Uniform Manifold Approximation and Projection (UMAP) plot of all 21,661 cells from 12-week-WT, 12-week-DB, 21-week-WT, and 21-week-DB mouse hindpaws. f Bar plot showing the number of cells for each cell type in each sample. Low-abundance clusters are re-plotted in the inset. g Dot plot showing the top 5 marker genes detected across all the 12 cell types. Data are mean ± SD; **/****p < 0.01, 0.0001, WT versus Leprdb/db, two-way ANOVA, Šidák / Mann–Whitney test

Single-cell analysis reveals hindpaw immune cell heterogeneity

Sequencing of CD45+ cells from 12- and 21-week-old WT and DB hindpaws generated an integrated dataset of 21,661 cells grouped into 12 clusters (Fig. 1c–f), defined as: macrophages (Pf4, C1qa, C1qc, Apoe, C1qb), type 2 conventional dendritic cells (cDC2) (CD74, H2-Aa, H2-Ab1, H2-Eb1, Mgl1), mast cells/basophils (M/B) (Mcpt2, Cma1, Cpa3, Mcpt4, Tpsb2), dermal γδ T (dTγδ) cells (Trdc, Cd163l1, Trdv4, Cxcr6, F2r), heterogeneous T (Thet) cells (Ikzf2, Ctla4, CD28, Trac, Icos), Type 2 innate lymphoid cells (ILC2) (Ctla2a, Gata3, Lmo4, Hlf, Il13), natural killer (NK) cells (Ccl5, xcl1, AW112010, Txk, Ly6c2), dendritic epidermal T cells (DETC; Cd7, Trat1, CD3e, Gem, Fermt2), type 1 conventional dendritic cells (cDC1) cells (Irf8, CD207, Tbc1d4, Cst3, Naaa), B cells (Igkc, Ighm, Ebf1, CD79a, Iglc2), monocyte-derived macrophages (MdMacro) (Plac8, Ifitm3, Lst1, Chil3, Thbs1), and neutrophils (Neut) (S100a9, S100a8, Retnlg, G0s2, Acod1) (Fig. 1g, Supplementary Fig. 1 and Supplementary Table 2) [30]. After quality control and filtering, the number of single-cell transcriptomes was 3,946 (12-week-WT), 5,413 (12-week-DB), 4,852 (21-week-WT), and 7,450 (21-week-DB). When each sample was represented as a proportion of the integrated cluster total, variation in cell numbers in each cluster was apparent (Fig. 1e, f). Macrophages were the most abundant cell type across all samples, while mast cell/basophil (M/B), dermal γδ T cell (dTγδ), heterogeneous T cell (Thet), and Type 2 innate lymphoid cell (ILC2) populations were substantially increased in 21-week-DB mice (Fig. 1f and Supplementary Table 3).

Gene expression changes in WT and DB mice

We investigated the functional features of the cells obtained from each group using GO biological process enrichment. We then compared 12-week-WT mice with 12-week-DB mice (Fig. 2a, b) and 21-week-WT mice with 21-week-DB mice (Fig. 2c, d; Supplementary Table 4). Comparing 12-week-DB mice with 12-week-WT mice identified T cell-related transcripts (e.g., Cd3e, Cd3g, Il2ra) upregulated in 12-week-DB mice, while transcripts typical of antigen-presenting cells (e.g., Cd74, H2-Aa/Ab1, C1qa) were downregulated. Consistent with these findings, the GO biological process enrichment was dominated by T cell development pathways (T cell differentiation, regulation of T cell receptor signaling, T cell activation), suggesting generalized T cell activation in 12-week-DB mice.

Fig. 2

Gene expression and pathway analysis: a Volcano plot for the 12-week-DB vs 12-week-WT comparison showing − log10 (p value) and log2 fold changes for all transcripts. Those upregulated in 12-week-DB in red, downregulated in 12-week-DB in blue. Gray genes = no statistically significant change in expression. b Ingenuity Pathway Analysis (IPA) of upregulated genes from the 12-week-DB compared with 12-week-WT. c) Volcano plot for the 21-week-DB vs 21-week-WT comparison showing − log10 (p value) and fold changes for all transcripts. Those upregulated in 21-week-DB in red, downregulated in 21-week-DB in blue. Gray genes = no statistically significant change in expression. d Pathway analysis for transcripts upregulated in 21-week-DB compared with 21-week-WT. e Venn diagram showing the overlapping of the gene expression from the comparison of 12-week-WT vs 21-week-WT and 12-week-DB vs 21-week-DB. f Pathway analysis showing the activated or inhibited pathways represented by the 975 differentially expressed genes unique to DB mice

Comparing 21-week-WT with 21-week-DB mice yielded a similar upregulation of T cell-related transcripts (e.g., Ctla2b, Gata3, Il13) and downregulation of proinflammatory innate immune responses (e.g., Ptgs2, Il1b, Mpeg1). However, comparison of 21-week-WT with 21-week-DB mice (Fig. 2c, d) revealed pathways related to RNA processing and gene expression. To further assess chronicity-associated shifts in gene expression in DB mice, we took the 1,289 genes differentially expressed between 12-week-DB and 21-week-DB mice and excluded 314 genes that were also differentially expressed in 12-week-WT versus 21-week-WT mice, resulting in 975 differentially expressed genes specific to DB mice (Fig. 2e, f). Many pathways upregulated in 12-week-DB or 21-week-DB mice spanned multiple cell types. Notably, IL-10, PD-1/PD-L1, and autophagy-related pathways were upregulated in 21-week-DB mice, while phagocytosis, Fc receptor, and B-cell/T cell-related signaling were downregulated.

Since our analysis showed a large variation in cell type-specific genes without a clear indication of the cell types responsible, we carried out cluster-specific comparative pathway analysis (Fig. 3). In age-matched WT versus DB comparisons, all samples showed disproportionate upregulation of pathways within the macrophage cluster, with DB mice showing more upregulation compared to WT mice of the same age (Fig. 3a). However, analysis of genes related to classical or alternative macrophage activation was inconclusive, reaffirming the heterogeneity of tissue macrophages (Fig. 3b, c).

Fig. 3

Comparative pathway analysis by cell type. a Heatmap showing comparative immune-specific pathway analysis for all cell types from 12-week-WT and 12-week-DB (left) and 21-week-WT and 21-week-DB (right) samples. b Dot plot showing the expression of macrophage classical activation and c macrophage alternative activation signaling genes

Cell–cell communication shows dysregulation of multiple signaling pathways

To generate more insight into the potential dysregulation of immune cell signaling in DB versus WT mice, we undertook an analysis of inferred signaling between the 12 leukocyte clusters across both timepoints (Supplementary Fig. 2). TGFβ is a pleiotropic cytokine with roles in adult tissue homeostasis and fibrosis [36]. In DB mice at 12 and 21 weeks of age, we observed a loss of B-cell-derived TGFβ signaling to macrophages, accompanied by a loss of monocyte-derived macrophage signaling to Type 2 innate lymphoid cells and heterogeneous T cells (Fig. 4a). At 12 weeks of age, we also saw an emergence of signaling related to the proinflammatory cytokine interleukin-1 (IL-1). Signaling from Type 1 classical dendritic cells to T cell-related clusters (natural killer cells, Type 2 innate lymphoid cells, heterogeneous T cells, and delta-gamma T cells) was detected in DB but not WT mice (Fig. 4b). There was also a notable absence of dendritic epidermal T cell-derived interleukin-4 (IL-4) signaling in 12-week-old DB mice, though Type 2 innate lymphoid cell-derived IL-4 signaling was retained (Fig. 4c). Neutrophil-derived complement signaling was also undetectable in 12-week-old DB mice (Fig. 4d). Finally, in 21-week-old DB mice, there was an emergence of macrophage-derived signaling via APRIL (“a proliferation-inducing ligand;” Fig. 4e) and galectin (Fig. 4f) signaling. Collectively, this indicates profound dysregulation of innate and adaptive immune cell signaling networks in DB skin at both ages tested.

Fig. 4

Cell-to-cell communication a Chord diagram showing the network of TGFβ signaling in 12-week-WT, 12-week-DB, 21-week-WT, and 21-week-DB. b IL-1 signaling in 12-week-WT and 12-week-DB. c IL-4 signaling in 12-week-WT and 12-week-DB. d Complement signaling in 12-week-WT and 12-week-DB. e APRIL signaling in 21-week-WT and 21-week-DB, and f galectin signaling in 21-week-WT and 21-week-DB

Role of macrophages in T2DM

Because of the disproportionate transcriptional upregulation of macrophages (Fig. 3a) and bidirectional changes in macrophage signaling associated with several key cytokines (Fig. 4), we next carried out unsupervised sub-clustering of the macrophage cluster. Unsupervised sub-clustering of macrophages revealed seven distinct clusters based on the expression of markers not consistent with known macrophage populations (Fig. 5a, b and Supplementary Table 5). To determine shifts in macrophage gene expression associated with chronicity of diabetes, we identified the 623 differentially expressed genes that were unique to DB mice (Fig. 5c). Pathway analysis of these genes (Fig. 5d and Supplementary Table 6) showed shifts in similar pathways compared to the comparison made across all cells (Figs. 2f and 5d), including downregulation of N-formyl-methionyl-leucyl-phenylalanine signaling in neutrophils, phagocytosis-related pathways, Fc receptor signaling and upregulation of IL-10, and phosphatase and tensin homolog (PTEN) signaling.

Fig. 5

Macrophage sub-clustering analysis. a UMAP plot of all 7699 macrophages grouped into 7 sub-clusters. b Dot plot showing the top five gene markers from each cluster. c Venn diagram showing the overlap of differentially expressed genes from 21-week-WT vs 12-week-WT and 21-week-DB vs 12-week-DB macrophages. d The pathway analysis of 623 genes unique to DB showing the activated or inhibited pathways from the 21-week-DB as compared to 12-week-DB

Lyve1 and MHCII cluster classification

A new classification system for resident tissue macrophages based on Lyve1 and MHCII expression was recently proposed [37, 38]. Our dataset shows that expression of Lyve1 is clearly delineated, with Lyve1 predominantly expressed in macrophage clusters 0 and 3 (Fig. 6a), and Lyve1+ cells representing 40–60% of the total macrophage population, depending on age/genotype (Fig. 6b). Interestingly, 12-week-DB mice had a slightly lower proportion of Lyve1+ macrophages than 12-week-WT mice, whereas 21-week-DB mice had approximately 50% more Lyve1+ macrophages than 21-week-WT. Pathway analysis of Lyve1+ vs Lyve1− macrophages showed that Lyve1 expression was associated with expression of genes related to mobility and vascular development, while Lyve1− macrophages were more dedicated to immune cell function (Fig. 6c). This is consistent with the findings of Krasniewski et al. and Chakarov et al., which suggest Lyve1+ macrophages are M2-like and Lyve1− macrophages are M1-like. This is further supported by the gene expression profile (Fig. 6d, e and Supplementary Table 7) which shows that Lyve1− macrophages express transcripts for antigen presentation (H2-Eb1, H2DMb1, H2-Ab1, CD74), and Lyve1+ macrophages express transcripts related to angiogenesis (Ang, Stab1, Cfh, Hspb1), a distinction also noted by Krasniewski et al. [37].

Fig. 6

Analysis of Lyve1+ and Lyve1− macrophage sub-populations: a UMAP plot showing Lyve1 expression across macrophage sub-clusters. b Bar plot showing the relative proportion of Lyve1+ and Lyve1− cells across samples. c GO biological process enrichment analysis of upregulated and downregulated genes from Lyve1+ vs Lyve1− macrophages. d Dot plot showing the expression pattern of Lyve1+ and e Lyve1−-specific genes across the samples

Layering MHCII expression onto the expression of Lyve1 permitted formation of four sub-populations [37]: Lyve1+ MHCIIhi, Lyve1+ MHCIIlo, Lyve1− MHCIIhi, and Lyve1− MHCIIlo (Fig. 7a, b). This categorization revealed an increase in the relative proportion of Lyve1+ MHCIIlo macrophages that was unique to 21-week-DB mice. In addition, the numbers of Lyve1+ MHCIIhi and Lyve1− MHCIIhi cells declined in 21-week-DB mice. This suggests a transition to an anti-inflammatory/angiogenic M2-like phenotype in 21-week-DB mice, whereas WT mice remain relatively stable in this regard as they age (Fig. 7b, c). Pathway enrichment for each sub-population showed distinct expression profiles. Lyve1+ MHCIIhi macrophages showed elevated expression of immune activation/inflammatory response genes; Lyve1− MHCIIhi cells increased the expression of genes related to cytokine responses and lymphocyte activation; Lyve1− MHCIIlo cells showed elevated expression of genes related to recognition of foreign organisms, and Lyve1+ MHCIILo cells expressed transcripts related to endocytosis and vascular development (Fig. 7d).

Fig. 7

Classification of macrophages into four sub-groups based on Lyve1 and MHCII expression. a UMAP projection showing the classification of macrophages based on the expression of Lyve1 and MHCII: Lyve1+/MHCIIhi, Lyve1+/MHCIIlo, Lyve1−/MHCIIhi, and Lyve1−/MHCIIlo. b Bar plot showing the relative proportion of the four sub-groups across the samples. c UMAP projection of four sub-groups for each sample. d GO biological process analysis of differentially expressed genes for each group

Histological characterization of DB mouse hindpaws

To better understand the distribution of Lyve1+ and MHCIIhi macrophages within the hindpaw, DB and WT tissues were stained with CD68/Iba1 and co-stained with Lyve1 (Fig. 8a, b) or MHCII (Fig. 8c, d) antibodies. Quantification of CD68/Iba1 immunofluorescence identified a significant increase in macrophage density in the hypodermis of 12-week-DB and 21-week-DB mice compared to their WT counterparts (Fig. 8e). This increase in macrophages was mirrored by a corresponding increase in the correlation between Lyve1 and CD68/Iba1 signal (Fig. 8f), consistent with the emergence of Lyve1+ macrophages in DB mice. However, there was no significant change in correlation between MHCII and CD68/Iba1 (Fig. 8g).

Fig. 8

Histological analysis of Lyve1+ macrophages in the hindpaws of 12- and 21-week-old aged WT and DB mice. Representative images of CD68/Iba1+ macrophages co-stained with a, b Lyve1 or c, d MHCII antibodies from the hindpaws of 12-week-WT, 21-week-WT, 12-week-DB, and 21-week-DB mice (n = 3; dotted white boxes = areas magnified in b and d. e Percent area quantifications for CD68/Iba1+ showed a significant increase in macrophage density in DB mice compared to WT groups. f This increase in macrophage density corresponded to a significant increase in Pearson’s correlation between CD68/Iba1+ signal and Lyve1+ signal for 12-week-DB and 21-week-DB mice compared to WT mice. g Quantification of MHCII stains showed no significant changes in Pearson’s correlation with CD68/Iba1+ signal. *p < 0.05: **p < 0.01. Data are mean ± SD (n = 3)

From an additional series of colocalization stains, we were able to detect a significant increase in the density of Lyve1+ cells in DB mice, which positively correlated with CD206. However, it should also be noted that despite Lyve1 having a somewhat negative correlation with CD68 alone, there was a significant increase in the correlation between CD68 and Lyve1 signals in 12- and 21-week-DB mice as compared to 12-week-WT mice (Fig. 9a–e). In contrast, there was only a moderate non-significant decrease in the percent area for MHCII+ cells in 12- and 21-week-DB hindpaws compared to the other groups (Fig. 9f–h), and MHCII had a negative correlation with both CD206 and CD68 (Fig. 9i, j). Alongside quantification and characterization for macrophages, we also determined whether an increase in T cell density was detectable, as the sequencing data suggested (Fig. 2). Immunofluorescent staining of CD3+ cells revealed a moderate but non-significant increase in small clusters of T cells within the dermis, suggesting a relatively small area of distribution (Supplemental Fig. 3).

Fig. 9

Histological characterization of Lyve1+ and MHCIIhi macrophages. a Representative image of Lyve1 and CD206 colocalization (white dotted box = the area magnified in the right-most image on each row). b Representative image of Lyve1 and CD68 colocalization. c Quantification of Lyve1+ percent area from co-staining with CD206 shows a significant increase in the percent area of Lyve1 in 21-week-DB mice as compared to all other groups. Pearson’s correlation for Lyve1 with d CD206 and e CD68 revealed a positive correlation between Lyve1 and CD206 and a significant increase in the correlation between Lyve1 and CD68 in the 12- and 21-week-DB mice as compared to 12-week-WT mice. f Representative image of MHCII and CD206 colocalization. g Representative image of MHCII and CD68 colocalization. h Quantification of MHCII percent area from co-staining with CD206 shows no significant differences between groups. Pearson’s correlations for MHCII with both i CD206 and j CD68 revealed a negative correlation between MHCII and CD206 and between MHCII and CD68, although there was a significantly higher colocalization between MHCII and CD68 within 21-week-WT mice as compared to 12-week-DB mice. *p < 0.05: **p < 0.01. Data are mean ± SD (n = 4–6)

In addition to verifying the Lyve1+ population as primarily CD206+, we also explored the extent to which the Lyve1+ or MHCII+ population showed close proximity to nerves or blood vessels, since it has been previously reported that both Lyve1+ and MHCIIhi macrophages are often in close proximity to CD31+ blood vessels and TUBB3+ nerves [37]. To this end, Lyve1 and MHCII were co-stained alongside PGP9.5 and CD31 to highlight nerves and endothelial cells, respectively. Lyve1+ cells reside predominantly in the deeper portions of the dermis, away from the nerve plexus which underlies the epidermis (Fig. 10a), while MHCIIhi cells have a strong association with the nerve plexus (Fig. 10b). In addition, we were able to identify Lyve1+ and MHCII+ cells adjacent to both large and small vessels of the foot (Fig. 10c, d). It should also be noted that we found a distinct population of MHCII+ cells in the superficial epidermis, (most likely epidermal Langerhans cells), and that this population also displayed a close association with intraepidermal nerve fibers (Fig. 10a).

Fig. 10

Histological association of Lyve1+ and MHCIIhi macrophages with nerves and blood vessels. Immunofluorescent staining for a Lyve1/PGP9.5 and b MHCII/PGP9.5 was used to identify associations of Lyve1+ and MHCII+ cells with the nerve plexus of the plantar skin (dotted white boxes show the region enlarged in the right column). MHCII+ cells showed a much closer association with the nerve plexus as compared to lyve1+ cells. A population of MHCII+ cells (putative epidermal Langerhans cells, green arrows) was identified in the epidermis. Immunofluorescent staining for c Lyve1/CD31 and d MHCII/CD31 was used to identify associations of Lyve1+ and MHCII+ cells with both large vessels (ovoid dotted line) and small vessels (dotted white box) of the hindpaw. Macrophages associated with larger vessels reside near the outer perimeter of the vessel, in the putative tunica externa [39]