Fig 2: Inhibitory effect of XN on LPS-binding to MD-2, TLR4 and CD14. Effect of increasing concentrations of XN (0–8 µg/mL) on LPS-binding to MD-2 (a), TLR4 (b) and CD14 (c) as well as receptor activity of hTLR4 HEK293 cells co-stimulated with LPS (100 ng/mL), XN (4 µg/mL) and sCD14 (1000 ng/mL) for 12 h (d). LPS, lipopolysaccharide; MD-2, myeloid differentiation factor 2; sCD14, soluble cluster of differentiation 14; TLR, toll-like receptor; XN, xanthohumol derived through a XN-rich hop extract. Data are expressed as means ± SEM. # = p < 0.05 compared to unstimulated control (0 ng/mL LPS, 0 µg/mL XN). * = p < 0.05 compared to LPS-stimulated cells.

Fig 3: Human alveolar macrophages express functional IFNLR1. Two-dimensional T-distributed stochastic neighbor embedding (t-SNE) plots were generated based on global gene expression relationships. IFNLR1 and two marker genes, the human alveolar macrophage (HAMs) marker CD163 and epithelial cell marker EPCAM, were displayed with level of intensity of blue (A). CD14 monocytes were immediately harvested. Macrophages were differentiated in GM-CSF for 7 days prior to harvest. HAMs were lavaged from transplant rejected lungs, plated overnight prior to harvest. We performed SDS PAGE and examined IFNLR1 expression, and the expression of monocyte (CD14) and macrophage (CD206, MHCII, CD68) markers in the three cell types. (B). HAMs were plated for 24h and subjected to flow cytometry analysis with antibodies again IFNLR1 and CD206. Top: Flow cytometry analysis of fluorescent intensity in unstained (red), phycoerythrin (PE) isotype incubated (black), and IFNLR1-PE incubated HAMs (blue). Bottom: IFNLR1 expression in CD206 positive cells (C). n=2 independent experiments (B, C).

Fig 4: Protein concentration of MD-2, TLR4 and CD14 in LPS-stimulated PBMCs obtained from healthy study participants. Representative blots (a) and densitometric analysis of MD-2 western blot (b), TLR4 protein concentration in total protein lysate (c) and sCD14 protein concentration in cell culture supernatant (d) of PBMCs obtained from study participants either receiving a placebo or XN stimulated with LPS (100 ng/mL) for 6 h. LPS, lipopolysaccharide; MD-2, myeloid differentiation factor 2; PBMC, peripheral blood mononuclear cell; sCD14, soluble cluster of differentiation 14; TLR, toll-like receptor; XN, xanthohumol derived through a XN-rich hop extract. Data are expressed as means ± SEM. * = p < 0.05.

Fig 5: An obese environment changes the macrophage phenotype in a survivin-dependent manner. a Gene expression analysis of THP-1-derived macrophages cultured with conditioned medium (CM) from visceral ASCs of lean donors (lnASCs) or subjects with obesity (obASCs). mRNA CM lnASC values are arbitrarily set to 1.0 (dotted line). n = 5 in each group, *p < 0.05; **p < 0.01 versus CM from lnASCs. b Western blotting of TGFß1, HIF-1a, CD14, PPAR? and survivin in THP-1-derived macrophages cultured with CM from lnASCs or obASCs. GAPDH was used as a loading control. Shown is a representative image and densitometry analysis (arbitrary units), n = 8 for survivin and n = 2–4 for other proteins. ***p < 0.001 versus CM from lnASCs. c Immunofluorescence of survivin in THP-1-derived macrophages cultured with CM from lnASCs or obASCs. Survivin is marked in red and the cell nucleus is marked in blue (DAPI). Shown is a representative image and fluorescence quantification. ***p < 0.001 versus CM from lnASCs. d Fluorescence and immunofluorescence analysis of internalized survivin in THP-1-derived macrophages co-cultured with HEK-293 cells. In the fluorescence images green denotes the GFP-tag of the adenovirus. In the immunofluorescence images, survivin is marked in red and the cell nucleus is marked in blue (DAPI). Shown is a representative image and fluorescence quantification. **p < 0.01 versus negative control; ***p < 0.001 versus negative control and ad-GFP. e Gene expression analysis in THP-1-derived macrophages co-cultured with HEK-293 cells overexpressing control adenovirus (ad-GFP) or survivin (ad-survivin). mRNA ad-GFP values are arbitrarily set to 1.0. n = 3, *p < 0.05; **p < 0.01 versus co-culture with ad-GFP. f Immunoblotting analysis of THP-1-derived macrophages cultured with CM-obASC with or without an anti-survivin neutralizing antibody. Shown is a representative image and densitometry analysis (arbitrary units). n = 3–4, *p < 0.05; **p < 0.01 versus CM-obASC without anti-survivin antibody (IgG). Data information: All values are expressed as mean ± S.E.M. Statistical analyses: unpaired Student’s t test or ANOVA with Tukey’s multiple comparisons test