Human intervention studies

All intervention studies were carried out in a randomized placebo-controlled cross-over design and in accordance with the ethical standards laid down in the Declaration of Helsinki of 1975 as revised in 1983. Studies were approved by the ethics committee of the University of Vienna, Vienna, Austria (reference numbers: 00367 and 00848) and were registered at clinical trials (NCT04847193, 16.03.2021; NCT06286644, 17.01.2024). The following exclusion criteria were defined for both studies: (1) following a special diet or (2) food malabsorption or (3) a history of diseases of the gastrointestinal tract or (4) the use of anti-inflammatory medication, whereas the following inclusion criteria were defined: normal weight (BMI > 18.5 kg/m2 or < 24.9 kg/m2), non-smoking people were enrolled after giving written informed consent and they should not have a viral or bacterial infection 3 weeks prior the study. In both studies, the participants were asked to refrain from hop-containing products two weeks prior to the studies and during the intervention.

Pilot study to determine time- and dose-response

To determine the optimal time of cell isolation after the ingestion of the IAA-rich hop extract and the optimal IAA doses, a single blinded, randomized pilot study was conducted in normal weight, healthy female volunteers. To prepare the study drink, which was prepared freshly right before the intake, 10 ml drinking water were mixed with thickener (Nestlé S.A., Vevey, Switzerland), sugar-free lemon flavour (SodaStream GmbH, Frankfurt am Main, Germany) and 0, 15, 45 or 90 mg IAA derived through an IAA-rich hop extract (Isohop®, generous gift from Barth-Haas Group GmbH & Co. KG Nuremberg, Germany). After an overnight fast and obtaining a blood sample, subjects were asked to consume the study drink within 15 min together with a standardised breakfast (2 medium-sized pretzels with 30 g butter). Blood was taken at 1, 2, 3, 4 and 6 h after consumption of the study drink. Between time point 4 and 6 there was another standardised meal. After the first study day and a 1-week wash-out phase, in which the participants again were asked to refrain from all hops containing foods and beverages, the intervention was repeated in a cross-over design until all four study days were completed. The study design and the procedure on the study day are summarized in Fig. 1a. Clinical parameters e.g. fasting glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cholesterol were determined in the serum and monocytes were isolated from the whole blood as detailed below.

Fig. 1

Effect of a single intake of different concentrations of an iso-alpha acids rich hop extract on the LTA-induced immune response of IL-6 protein concentration in monocytes of healthy study participants over time. a Study design and study day of the time- and dose-response study, b protein concentration of IL-6 in cell culture supernatant of monocytes stimulated with 10 µg/ml LTA for 6 h isolated from healthy female study participants of the time- and dose-response study receiving either a placebo or IAA rich hop extract in different concentrations (0 mg, 15 mg, 45 mg or 90 mg). Data are presented as means ± SEM, n = 5. The mean of IL-6 protein concentration of LTA-stimulated monocytes from fasting blood (T0) of all intervention groups are shown in one column. IAA: iso-alpha acids, IL: interleukin, LTA: lipoteichoic acid

Assessment of the acute effects of the IAA-rich hop extract

In a second study which was based on the results of the time- and dose-response study, the effect of 15 mg IAA derived through an IAA-rich hop extract compared to a placebo were assessed. Male and female participants were randomly assigned in a single-blinded design to receive a study drink as described before, containing 0 or 15 mg of IAA derived through an IAA-rich hop extract. The study drink was consumed within 15 min together with a standardised breakfast (2 medium-sized pretzels with 30 g butter). The participants were fasted overnight and blood samples were taken before the study drink was consumed and 1 h after consumption of the study drink. After the first study day and a 1-week wash-out phase, in which the participants were again asked to refrain from hops-containing foods and beverages, the intervention was repeated in a cross-over design. The study design and the procedure on the study day is summarized in Fig. 2a. Clinical parameters were determined in the serum and PBMCs were isolated from whole blood as detailed below. Demographic characteristics of the study subjects are reported in Tables 1 and 2.

Fig. 2

Effect of a single intake of 15 mg of an iso-alpha acids rich hop extract on the LTA-induced immune response of proinflammatory cytokines in PBMCs of healthy study participants. a Study design and study day of the assessment of the acute effects of IAA, protein concentrations of (b) IL-6 and (c) IL-1ß in cell culture supernatant of PBMCs stimulated with 0–10 µg/ml LTA for 24 h and 48 h isolated from healthy study participants of the assessment of the acute effects of IAA receiving either a placebo or 15 mg IAA rich hop extract. Data are presented as means ± SEM, n = 13. *p < 0.05. IAA: iso-alpha acids, IL: interleukin, LTA: lipoteichoic acid, n.d.: not detectable, PBMC: peripheral blood mononuclear cell

Table 1 Characteristics of healthy female subjects enrolled in the dose- and time- response studyTable 2 Characteristics of healthy subjects enrolled in the study to assess the acute effect of IAA on LTA-dependent immune response of PBMCsIsolation of monocytes

Monocytes were isolated from whole blood samples collected from participants of the time- and dose-response study or from healthy, naïve, normal weight donors. After obtaining written informed consent and an overnight fast, blood was drawn from healthy, normal weight donors, with approval from the ethics committees of the University of Vienna, Vienna, Austria (reference number 00586). Monocytes were isolated from whole blood samples as detailed before using a density gradient centrifugation and the pluriSelect system following the instructions of the manufacturer (pluriSelect Life Science UG & Co. KG, Leipzig, Germany). In brief, blood was layered onto monocytes density gradient media (1.068 g/ml) in separation tubes (pluriSelect Life Science UG & Co. KG, Leipzig, Germany) and centrifuged at room temperature for 15 min. Cells were collected and used for further experiments as described below.

Isolation of PBMCs

PBMCs were isolated from whole blood samples as described in detail before [11]. In brief, cells were isolated by density gradient centrifugation. In brief, blood was layered onto Pancoll solution (1.077 g/ml) and centrifugated for 30 min at room temperature. Cells were collected and used for further experiments as described below.

Stimulation of monocytes and PBMCs obtained after an oral ingestion of iso-alpha acids derived through an IAA-rich hop extract

Isolated monocytes and PBMCs were cultivated in RPMI-1640 medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach, Germany) and 100 µg/ml streptomycin and 100 U/ml penicillin (Pan-Biotech GmbH, Aidenbach, Germany) at 37 °C in a humidified 5% CO₂ atmosphere for 1 h. Subsequently, cells were either challenged with 0–10 µg/ml LTA for 6 h in the pilot study and for 24 and 48 h in the assessment of the acute effects study, respectively. Cell culture supernatant was collected at the end of the experiment and stored at − 80 °C until further use.

Experiments in monocytes isolated from naïve healthy donors

Isolated monocytes were cultivated in RPMI-1640 medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach, Germany) and 100 µg/ml streptomycin and 100 U/ml penicillin (Pan-Biotech GmbH, Aidenbach, Germany) at 37 °C in a humidified 5% CO₂ atmosphere for 1 h. Subsequently, cells were preincubated for 1 h with different concentrations of iso-alpha acids (0.3125–1.25 µg/ml) and then treated with 10 µg/ml LTA for 6 h. Cell culture supernatant was collected at the end of the experiment and stored at − 80 °C until further use.

Cell culture experiments with hTLR2 transfected HEK cells

A commercially available reporter gene assay with TLR2 transfected HEK293 cells was used to assess the effects of iso-alpha acids on TLR2 ligand concentration (InvivoGen, Toulouse, France). The transfected human TLR2 cells were grown according to the instructions of the manufacturer. In brief, cells were grown in a humidified, 5% carbon dioxide atmosphere using DMEM media (Pan-Biotech GmbH, Aidenbach, Germany) containing 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach, Germany) and 100 µg/ml streptomycin and 100 U/ml penicillin (Pan-Biotech GmbH, Aidenbach, Germany). After reaching 80% confluence, cells were challenged with 0–10 µg/ml LTA in the presence of 0–40 µg/ml iso-alpha acids dissolved in HEK-Blue™ Detection medium (InvivoGen, Toulouse, France) for 12 h. Colour changes of the medium being indicative of ligand concentration and binding were determined at 655 nm.

Cell culture experiments with J774A.1 cells

J774A.1 cells (DMSZ, Braunschweig, Germany) showing a morphology similar to macrophages/monocytes, were cultured in Dulbecco´s Modified Eagle Medium (Pan-Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach Germany), 100 µg/ml streptomycin and 100 U/ml penicillin (Pan-Biotech GmbH, Aidenbach, Germany) at 37 °C in a humidified 5% CO₂ atmosphere. At 80% confluency, cells were preincubated for 1 h with or without 5 ng/ml anisomycin (Biomol GmbH, Hamburg, Germany), afterwards they were preincubated for 1 h with iso-alpha acids (0–25 µg/ml) and then treated with 10 µg/ml LTA for 6 and 24 h. The supernatant was collected at the end of the experiment and stored at − 80 °C until further use.

Enzyme-linked immunosorbent assays (ELISA´s)

IL-6 and IL-1ß protein concentrations were analysed in cell culture supernatant of monocytes and PBMCs or J774A.1 cells using commercially available DuoSet® ELISA Development Systems (R&D®, Minneapolis, USA) kits.

Western blot analysis

J774A.1 cells were homogenized in RIPA buffer (20 mmol/l 3 [N-morpholino] propanesulfonic acid, 150 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid, 1% Nonidet P-40, and 0.1% sodium dodecyl sulfate) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) to obtain protein lysates. Protein lysates (5 µg/lane) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were further incubated with specific primary antibodies (phosphorylated c-Jun N-terminal kinase (JNK) and total JNK; phosphorylated p38 MAPK and p38 MAPK; Cell Signaling Technology, MA, USA) and the respective secondary antibody (anti-rabbit IgG, HRP-linked; Cell Signaling Technology, MA, USA). To detect the protein bands, Clarity Western Enhanced Chemiluminescence Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA) were used. Densitometric analyses of detected bands were performed with ImageLab (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analyses

Data are presented as means ± standard error of the means (SEMs). Grubb´s test was performed before statistical analysis to identify outliers. Homogeneity of variances was tested and data were log-transformed if data were not normal distributed or in case of inhomogeneity of variances before performing further statistical tests. A paired t-test was used to determine statistically significant differences between interventions and to analyse differences between C- and LTA-stimulated cells of Western Blot analysis. One-way ANOVA was used for all other comparisons (GraphPad Prism Software, CA, USA). A p-value < 0.05 was defined as threshold for statistical significance.