Figure 7. CD4⁺ T cells in reaction with 10-HOME polarize macrophages to M1 phenotype.

(A) Gene interaction networks from human bulk RNA-Seq. Upregulated genes in red and downregulated are in green, in log₂ fold change. Table exhibited in Supplemental Figure 21A. (B) Increased expression of CD38 (M1 macrophage marker) in 10-HOME–treated mice compared with vehicle. Murine mammary fat pads stained with anti-CD38 antibody (DAB) and nucleus (hematoxylin). The cells in adipose tissues are bordered by adipose cells. Cells coexpressing DAB and hematoxylin were considered for analysis (arrows). Data presented as mean ± SEM, vehicle (n = 7) and 10-HOME (n = 7) mice. Scale bar: 100μm. Enlarged image in Supplemental Figure 22. (C) No significant difference of CD163 (M2 macrophage marker) in 10-HOME treated mice. Murine mammary fat pads stained with anti-CD38 antibody (DAB). Cells co-expressing DAB and hematoxylin were considered for analysis (arrows). Data presented as mean ± SEM, vehicle (n = 7) and 10-HOME (n = 7) mice. Scale bar: 100μm. Enlarged image in Supplemental Figure 23. (D) Quantification of B–C. Total number of CD38⁺ and CD163⁺ cells were calculated. Data presented as mean ± SEM, (n = 7). Mann Whitney test was used to determine vehicle versus 10-HOME CD38 (P = 0.0006) and CD163 (P = 0.558). (E) Schematic representation of macrophage/T cell transwell assay. PBMC-derived naive T cells treated with 10-HOME (blue), PBMC-derived M0 macrophages (green). (F) Increased polarization to M1 phenotype (CD86-human M1 marker) in the transwell coculture of PBMC-derived M0 macrophages incubated with10-HOME–treated naive CD4⁺ T cells. Flow cytometry analyses with anti-CD86 (FITC) and anti-CD14 (PE). Representative plots: vehicle treated and 10-HOME treated, histograms of cells with isotype control for CD86. Data presented as mean ± SD, (n = 8). Mann Whitney test was used for comparison of vehicle versus 10-HOME (P = 0.0152). (G) Unaltered M2 phenotype(CD163). Flow cytometry analyses with anti-CD163 (APC) and anti-CD14 (PE). Representative plots, vehicle treated and 10-HOME-treated, histograms with isotype control for CD163. Data presented as mean ± SD, (n = 8). Mann Whitney test was used to determine vehicle versus 10-HOME (P = 0.0749).