Single-cell transcriptomics reveals variations in monocytes and Tregs between gout flare and remission

Abstract

Gout commonly manifests as a painful, self-limiting inflammatory arthritis. Nevertheless, the understanding of the inflammatory and immune responses underlying gout flares and remission remains ambiguous. Here, based on single-cell RNA-Seq and an independent validation cohort, we identified the potential mechanism of gout flare, which likely involves the upregulation of HLA-DQA1+ nonclassical monocytes and is related to antigen processing and presentation. Furthermore, Tregs also play an essential role in the suppressive capacity during gout remission. Cell communication analysis suggested the existence of altered crosstalk between monocytes and other T cell types, such as Tregs. Moreover, we observed the systemic upregulation of inflammatory and cytokine genes, primarily in classical monocytes, during gout flares. All monocyte subtypes showed increased arachidonic acid metabolic activity along with upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2). We also detected a decrease in blood arachidonic acid and an increase in leukotriene B4 levels during gout flares. In summary, our study illustrates the distinctive immune cell responses and systemic inflammation patterns that characterize the transition from gout flares to remission, and it suggests that blood monocyte subtypes and Tregs are potential intervention targets for preventing recurrent gout attacks and progression.

Keywords: Immunology; Inflammation; Monocytes; T cells.

Figure 1. Single-cell transcriptome profiling of PBMCs between gout flare and remission.

(A) Flowchart of the overall experiment design. Two paired peripheral blood samples (6 in total) were collected from each of the same 3 patients with gout flare (1 sample/patient), and later during gout remission (1 sample/patient). The samples were dissociated into single cells and sorted for scRNA-Seq. (B) The t-SNE representations of integrated single-cell transcriptomes for the 34,736 PBMCs (n = 6), grouped by disease (left), and cell types (right). (C) Heatmap of all identified clusters after dimensional reduction. The top 10 marker genes colored by their expression level were used for downstream analyses. Clusters were reordered into respective cell types. (D) Bar plot showing cell fractions of leukocyte subtypes in patients with gout flare and gout remission, color-coded for the different cell types identified in this study. (E) Expression levels of canonical cell markers used to identify cell types. Feature plot represented by color gradient, with low expression depicted by gray and high expression represented by red.

Figure 2. Functions of each monocyte subtype that contribute to immune responses in patients with gout flare and remission.

(A) The t-SNE representations of integrated single-cell transcriptomes for the 7,499 myeloid cells (n = 6), color-coded by disease (left), and cell types (right). (B) Expression levels of canonical cell markers used to label clusters. Dot plot represented by color gradient, with low expression depicted by blue and high expression shown in red. (C) Bar plot of cell fractions for myeloid cell subtypes stratified by groups. (D) Heatmap of the DEGs among monocyte subtypes between gout flare and gout remission. The heatmap is colored by average log(FC). All displayed genes are statistically significant at P<0.05. (E) t-SNE plots illustrating the expression of characteristic cytokine markers in monocyte subtypes. Feature plot represented by color gradient, with low expression shown in gray and high expression depicted in red. (F) Heatmap of the AUC score t values for the expression regulation by transcription factors of monocytes subtypes, as estimated using SCENIC. (G) The regulon-specific AUC score t values for the expression regulation by transcription factors of the monocyte subtypes, as estimated by SCENIC.

Figure 3. HLA-DQA1^hi nonclassical monocytes regulate monocyte subtype differentiation in gout flare.

(A) KEGG pathway enrichment analysis for classical, intermediate, and nonclassical monocytes (left to right) was performed using the upregulated genes. (B) Single-cell trajectory analysis integrating cluster information. (C) Dynamics of DEGs (P_adj of Wilcoxon’s rank-sum test < 0.05, log₂(FC) > 1) between classical and nonclassical monocytes. (D) Representative box plots depicting the expression of HLA-DQA1 in classical and nonclassical monocytes during gout flare and remission. Data presented as median with IQR. The box represent the IQR, which spans from the lower to the upper quartile, while the box whiskers indicate the range of the data, excluding any outliers. Outliers are represented by individual points beyond the whiskers and are defined as values that fall outside the threshold 1.5 times the IQR range. Data from 21 patients with acute gout flare and 23 patients with gout remission are shown. Statistical analysis was performed by 2-tailed Student’s t test (*P < 0.05).

Figure 4. The role of CD4⁺ T cells subtypes in gout flare and remission.

(A) t-SNE representations of T/NK clusters from gout flare and remission. (B) Dot plot for the expression of canonical marker genes in all T cell and NK cell subtypes. (C) The proportions of cell subtypes in gout flare and remission. (D) Heatmap of DEGs in CD4⁺ T cells based on pairwise comparisons between gout flare and remission. (E) The t-SNE plots depict the expression of characteristic cytokines in CD4⁺ T cells. (F) The percentage of T, CD4⁺, Treg, Th1, Th2, and Th17 T cells in gout flare (n = 41) and remission (n = 35). Data presented as median with IQR. The box represent the IQR, while the whiskers indicate the data range without outliers. Outliers are shown as individual points beyond the whiskers and are defined as values outside the 1.5 times the IQR range threshold. Statistical analysis performed by 2-tailed Student’s t test (**P < 0.01). (G–I) Suppressive assay of Tregs (data shown as mean ± SD; n = 4 independent experiments). (G) The proliferation index by coculture of eFluor 450 dye–labeled autologous Teffs with autologous Tregs isolated from patients with gout flare and those with gout remission. Statistical analysis was performed by 2-way ANOVA (*P < 0.05). (H) The production of IFN-γ and IL-10 in cell culture supernatant of Tregs isolated from patients with gout flare and gout remission. Statistical analysis was conducted by 2-tailed Student’s t test. (I) The production of IFN-γ and IL-10 from the coculture of eFluor 450 dye–labeled autologous Teffs and autologous Tregs, isolated from patients with gout flare and gout remission. Statistical analysis performed by 2-way ANOVA (*P < 0.05).

Figure 5. CellChat analysis highlighting the intercellular communication between acute gout flare and remission.

(A) Comparison of the inferred interaction number (left) and interaction strength (right) in gout flare and remission. (B and C) Dot plots of the inferred interaction number (B) and interaction strength (C) between acute gout flare and remission. Blue lines indicate that the displayed communication is increased in gout remission, whereas red lines indicate its increase during gout flare. (D) Comparison of the significant ligand/receptor pairs between gout flare and remission, which contribute to signaling from classical monocytes (CM) and nonclassical monocytes (NCM) to DCs, intermediate monocytes (IM), and T cells (TC), including Treg, KLRB1⁺CD4⁺ TC, cytotoxic CD8⁺ TC, and B cells (BC). Dot color reflects communication probabilities, and dot size represents the computed P values. The empty space indicates a communication probability of zero. The P values were calculated based on 1-sided permutation test.

Figure 6. Metabolic properties of monocyte subtypes between acute gout flare and remission.

(A) The heatmap of significantly altered metabolic pathways for all PBMCs between gout flare and remission. (B) Violin plots of selected marker genes (upper row) related to the arachidonic acid pathway for multiple cell subpopulations. The left column presents the cell subtypes identified based on combinations of marker genes. (C) Violin plots for the average expression of genes related to the arachidonic acid pathway in each monocyte subtype between acute gout flare and remission. The P values were calculated using a 2-sided Wilcoxon rank-sum tests. Data are from single-cell transcriptomes of 3 independent patients with gout (**P<0.01, ***P<0.001). (D and E) Heatmaps of the significantly altered metabolic pathways in monocyte subtypes (D) and all PBMCs (E) between gout flare and remission.