Fig. 6: Mincle receptor is increased and interacts with Fu-Hp.

a The Venn diagram illustrating the number of genes with significantly increased expression (p value < 0.05) involved in the innate immune response pathway, across three cohorts (Seoul, GSE232753, and GSE65682) and increased genes in the macrophage population of scRNA-seq data. Two-sided p-values were calculated using the likelihood ratio test and adjusted for multiple comparisons using the Benjamini-Hochberg method to control the false discovery rate (FDR). b, Box-and-whisker plot showing the expression of the CLEC4E gene across three cohorts: Seoul (HCs = 12; SPs = 24), GSE232753 (HCs = 8; SPs = 20), and GSE65682 (HCs = 42; SPs = 479). The lower and upper hinges of the box represent the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values within 1.5 times the interquartile range. The median value is depicted by the line within the box. Statistical significance was calculated with a two-tailed t-test without adjustment (*p < 0.05; ***p < 0.001). c The violin plot depicting expression levels of CLEC4E gene in macrophage and monocyte population from PBMCs with or without Fu-Hp treatment. P values were calculated by the FindMarkers function in Seurat 5.0.1 package using the default two-sided Wilcoxon Rank Sum test (****p < 0.0001). d The violin plot displaying the expression levels of CLEC4E gene in subpopulations of monocytes (2, 19, and 27) and macrophages (7, 18, and 29). e The relative expression of CLEC4E gene in human primary monocytes after treatment with HC-Hp or Fu-Hp for indicated time point using qRT-PCR (n = 4 per group). Statistical significance was calculated with a two-sided unpaired t-test with Sidak-Bonferroni test and presented as means ± SD. arb. units, arbitrary unit. f, Immunoprecipitation assessing the interaction between HA-tagged Mincle and Hp purified from HCs and SPs (n = 4 per group). g Quantification of β-Hp pulldown by HA-Mincle (n = 4 per group). The graph showing the relative intensity of β-Hp bands normalized to HA-Mincle bands. Statistical significance was calculated with an unpaired t-test with Mann-Whitney test and presented as means ± SEM (*p < 0.05; **p < 0.01). h Schematic illustration of examining the interaction using TIRF microscopy. i, TIRF microscopy images of SNAP-Mincle and ATTO488-Hp. HeLa cells expressing SNAP-Mincle were labeled with a membrane-impermeable SNAP dye (red) and incubated with ATTO488-labeled HC-Hp or Fu-Hp (green). White arrowheads indicate colocalization of Mincle and Hp. Scale bars, 10 μm (right) or 0.1 μm (left). j, The bar plot showing the quantitation of colocalization of β-Hp with Mincle (n = 5). Statistical significance was calculated with a two-tailed unpaired t-test and presented as means ± SD (****p < 0.0001). k,l MST analysis assessing the interaction between HA-tagged Mincle and Hp purified from different cohorts. MST (left) traces and (right) dose response curves were shown. The left panel shows the representative MST trace corresponding to the titration of Hp, and the right panel indicates changes in thermophoresis fitted to yield a KD of 22.9 ± 5.15 μM (k) and a KD 5.7 ± 1.01 μM (l). Data are presented as mean ± standard deviation.