Fig. 3

Saturated VLCFA exposure initiates pro-inflammatory chemokine production in human macrophages. A Human macrophages derived from healthy control donors (n = 14) were treated with different concentrations of C26:0 or LPS for 24 h before expression of the pro-inflammatory cell surface marker CD86 was assessed by flow cytometry. B Human healthy control macrophages (n = 4–6) were treated with C26:0 (100 µM) or the solvent EtOH for the indicated time. RT-qPCR was carried out to measure mRNA levels of CXCL8, CCL3 and CCL4, and normalized to HPRT1. C Healthy control macrophages (n = 4) were treated with C26:0 (100 µM) or the solvent EtOH for 24 h. Supernatants were analysed for CXCL8, CCL3 and CCL4 protein levels using Luminex ELISA bead assays. The heat map indicates fold changes to solvent-treated samples. D RT-qPCR of CXCL8, CCL3 and CCL4 mRNA levels were normalized to HPRT1 in macrophages (n = 4) treated with either C16:0 (100 µM) or C26:0 (100 µM) for 12 or 24 h. E RT-qPCR of CXCL8 expression in healthy control macrophages (n = 3) treated with C26:0 (100 µM) or the CD36 inhibitor (CD36i) sulfosuccinimidyl oleate (100 µM) or with both compounds for 24 h. F RT-qPCR of MMP9, MMP14 and PLAUR normalized to HPRT1 was performed in healthy control macrophages (n = 5) treated with either C16:0 (100 µM) or C26:0 (100 µM) for 24 h. G Healthy control macrophages derived from 4 donors were treated with C26:0 (100 µM) or the solvent EtOH for 24 h before podosome structures were visualized by staining f-actin with AlexaFluor⁴⁸⁸-phalloidin and the frequency of podosome-positive macrophages being determined by fluorescence microscopy. One-way ANOVA and Fisher’s LSD multiple comparison test were performed in A and the ratio paired t-test was used in B, D–G: **p < 0.01; *p < 0.05; ns not significant. Boxplots indicate median ± interquartile range, while whiskers show minimum and maximum. Bar graphs show means ± standard deviations