A Xanthohumol-Rich Hop Extract Diminishes Endotoxin-Induced Activation of TLR4 Signaling in Human Peripheral Blood Mononuclear Cells: A Study in Healthy Women

Abstract

Infections with Gram-negative bacteria are still among the leading causes of infection-related deaths. Several studies suggest that the chalcone xanthohumol (XN) found in hop (Humulus lupulus) possesses anti-inflammatory effects. In a single-blinded, placebo controlled randomized cross-over design study we assessed if the oral intake of a single low dose of 0.125 mg of a XN derived through a XN-rich hop extract (75% XN) affects lipopolysaccharide (LPS)-induced immune responses in peripheral blood mononuclear cells (PBMCs) ex vivo in normal weight healthy women (n = 9) (clinicaltrials.gov: NCT04847193) and determined associated molecular mechanisms. LPS-stimulation of PBMCs isolated from participants 1 h after the intake of the placebo for 2 h resulted in a significant induction of pro-inflammatory cytokine release which was significantly attenuated when participants had consumed XN. The XN-dependent attenuation of proinflammatory cytokine release was less pronounced 6 h after the LPS stimulation while the release of sCD14 was significantly reduced at this timepoint. The LPS-dependent activation of hTLR4 transfected HEK293 cells was significantly and dose-dependently suppressed by the XN-rich hop extract which was attenuated when cells were co-challenged with sCD14. Taken together, our results suggest even a one-time intake of low doses of XN consumed in a XN-rich hop extract can suppress LPS-dependent stimulation of PBMCs and that this is related to the interaction of the hop compound with the CD14/TLR4 signaling cascade.

Keywords: CD14; LPS; TLR4; hop; inflammation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Fluorescence imaging and XN determination of PBMCs isolated from buffy coat of healthy donors co-cultured with Caco-2 cells incubated with XN. (a) Graphical illustration of the experimental co-culture setup using Caco-2 cells and PBMCs. (b) Representative pictures of fluorescence of XN in PBMCs cells after incubation of Caco-2 cells ± XN (0.125 mg/mL derived through a XN-rich hop extract) for 1 h (magnification 400×) in a co-culture model. White arrows indicate autofluorescence of XN. PBMC, peripheral blood mononuclear cell, XN, xanthohumol. Data are expressed as means ± SEM.

Figure 2

Graphical visualization of the study design. (a) Study design and (b) the procedure performed on each day of the study. XN, xanthohumol derived though a xanthohumol-rich hop extract.

Figure 3

Cytokine concentrations in supernatant of LPS−stimulated PBMCs obtained from healthy study participants. Protein concentrations of IL−−1β (a), IL−−6 (b) and TNF−−α (c) in cell culture supernatant of PBMCs stimulated with 0 or 100 ng/mL LPS for 2 h isolated from healthy study participants receiving either a placebo or the study drink containing XN derived through a XN−rich hop extract. IL, interleukin; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; XN, xanthohumol. Data are expressed as means ± SEM. * = p < 0.005.

Figure 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.

Figure 5

Receptor activities of HEK293 cells co-stimulated with LPS and XN for 12h. HEK293 cells were stimulated with LPS (100 ng/mL) and increasing concentrations of XN (0–8 μg/mL) for 12 h. LPS, lipopolysaccharide; XN, xanthohumol derived through a XN-rich hop extract. Data are expressed as means ± SEM. a = p < 0.05 compared to 0 XN + LPS, b = p < 0.05 compared to 0.5 XN + LPS, c = p < 0.05 compared to 1 XN + LPS, d = p < 0.05 compared to 2 XN, e = p < 0.05 compared to 4 XN + LPS.

Figure 6

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.