Figure 5. Wnt signaling activation limits innate immune exhaustion phenotype and DNA methylation alterations observed in BMMCs under repetitive LPS stimulation.

(A) scRNA-seq UMAP feature plots for transcription factor TCF7L2 motif enrichment and gene expression. Numbers indicate different cell clusters, as outlined in Figure 2A.

(B) RT-qPCR for key exhaustion genes in BMMCs under PBS control or repetitive LPS stimulation in the presence of different concentrations of Wnt agonist 1. Expression levels were normalized to the geometric mean of Ube2l3, Oaz1, and Nktr (mean expression ± SD; n = 3–6; one-way ANOVA with Sidak’s multiple comparisons test; ****p-adj < 0.0001; ***p-adj < 0.001; **p-adj < 0.01; *p-adj < 0.05; ns, not significant; differences are not significant unless otherwise specified; see Table S8 for exact p values).

(C) Flow cytometry mean fluorescence intensity (MFI) for exhaustion (CD38, MARCO, PD-L1, CXCR2) and macrophage (F4/80) markers relative to PBS control. Boxplots indicate median MFI values (n = 5–7; one-way ANOVA with Sidak’s multiple comparisons test).

(D) Gating strategy (top) and population statistics (bottom) for non-classical (Ly6C^low), intermediate (Ly6C^int), and classical (Ly6C^high) monocyte subtypes in cultured BMMCs (n = 6–13; one-way ANOVA with Sidak’s multiple comparisons test).

(E) NAD⁺ levels in cultured BMMCs normalized to total protein levels (n = 3–6; one-way ANOVA with Sidak’s multiple comparisons test).

(F) Correlation heatmap for average DNA methylation levels at key exhaustion loci in cultured BMMCs (n = 3–9 for each condition).

(G) UCSC browser track views of average DNA methylation at the Plac8 promoter (chr5: 100,572,230–100,572,607), Cebpa/g distal enhancer (chr7: 35,064,805–35,065,304), and Tcf7l2 intron (chr19: 55,768,986–55,769,585). ENCODE annotated promoters (red) and enhancers (orange) are depicted in the bottom track.