Imukin

According to previous publication in human sepsis, a dose of 100mcg of IFNγ (Imukin®, Boehringer, Ingelheim, Germany) was subcutaneously injected for 3 to 5 days (cohort 1) or on days 2–4–7-9-11 (cohort 2) as reported [6 (link), 9 (link), 18 (link), 19 (link)]. The treatment was repeated to reach an increased mHLA-DR expression above 8000 AB/C (cohort 1) or MFI > 20 in cohort 2 (low threshold fixed at MFI 20). Clinical tolerance and systemic or local (injection site) symptoms of inflammation were carefully checked.

Whole blood of patient and controls was diluted 1:5 in RPMI into 96-well F plates (Corning) and activated by single stimulation with phytohemagglutinin (PHA, 10 μg/ml; Sigma-Aldrich) or LPS (1 μg/ml, List Biochemicals) alone or by co-stimulation with IFN-γ (2 × 10⁴ IU/ml, Imukin, Boehringer Ingelheim), and supernatants were taken after 24 h incubation at 37 °C/5%CO₂. Cytokines were measured by multiplexed bead array (IFN-γ, TNF-α, IL-12, IL-10, IL-6, IL-17A, IL-1β, R+D Systems Fluorokinemap) on a Luminex analyzer (Bio-Plex, Bio-Rad, UK).

Whole blood samples were collected from healthy controls and the patients into heparin-containing collection tubes. Samples were diluted 1:2 in RPMI 1640 supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin (#15,140,122, Thermo Fisher Scientific). Samples were either incubated with medium alone, alone with BCG (Mycobacterium bovis-BCG, Pasteur substrain) at a MOI of 20, or with BCG plus human recombinant (rh) IL-12 (20 ng/ml; #219-IL-005/CF, R&D Systems), BCG plus IFN-γ (Imukin, Boehringer Ingelheim) for 48 h at 37 °C and 5% CO₂. The supernatants were collected and assessed for their IL-12p40 (#DP400, R&D Systems) and IFN-γ (#DIF50, R&D Systems) content in accordance with the manufacturer’s protocol.

Whole blood was diluted 1:5 in RPMI into 96-well F plates (Corning) and activated by single stimulation with IL-12 (20 ng ml⁻¹; R&D Systems), PHA (10 μg ml⁻¹; Sigma-Aldrich), LPS (1 μg ml⁻¹) List Biochemicals, IFN-γ (2 × 10 IU ml⁻¹, Imukin, Boehringer Ingelheim), IFN-α (2 × 10³ IU ml⁻¹, Intron A, Schering Plough, UK) or using co-stimulations as indicated. Supernatants were taken at 24 h. Cytokines were measured using standard ELISA according to the manufacturer's recommendations (IFN-γ, Pelikine, Sanquin, NL), or multiplexed (TNFα, IL-12, IL-10, IL-6, R+D Systems Fluorokinemap) on a Luminex analyser (Bio-Plex, Bio-Rad, UK). Data were statistically analysed by the two-tailed Mann–Whitney test using Prism 6 (GraphPad Software).

Whole-blood samples from one healthy control, one healthy travel control, P1 and P2 were collected in heparin-containing collection tubes. Samples were diluted 1:2 in RPMI 1640 supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific). Stimulation was performed in a 48-well plate. Briefly, 1 mL of whole-blood was used per well and per set of conditions. Samples were incubated with medium alone, with live BCG (M. bovis-BCG, Pasteur substrain) at a MOI of 20, or with BCG plus IL-12 (20 ng/mL; #219-IL, R&D Systems), or BCG plus IFN-γ (Imukin, Boehringer Ingelheim) for 48 hours at 37°C under an atmosphere containing 5% CO₂. The supernatants were collected and used for ELISA. Data were compared to published findings for healthy and travel controls.^{61 (link)}

The herein described macrophage-neutrophil co-culture assay was adapted from Marwick et al. [24 (link)]. Macrophage differentiation was induced by cultivation of isolated monocytes in RPMI 1640 medium supplemented with GlutaMAX™, 10% FCS and 100 ng/ml M-CSF (130-096-491, Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 days, with medium change performed on the third day. One day before the co-culture experiment, monocyte-derived macrophages (MDMs) were harvested and re-seeded in 48-well-plates at 1 × 10⁵ cells per well. To induce a pro-inflammatory phenotype, MDMs were stimulated with 20 ng/ml interferon-gamma (INFy) (Imukin®) (Boehringer Ingelheim, Vienna, Austria). On the day of co-culture, MDMs were washed twice with 1 × DPBS, before autologous neutrophils were added at a 1:5 or 1:10 ratio (MDMs:neutrophils) in RPMI 1640 medium, without FCS and M-CSF. In indicated experiments, neutrophils were cultured for 24 h before co-culture assays in order to obtain apoptotic neutrophils. After 30 min of co-culture at 37 °C, cells were stimulated with 1 ng/ml lipopolysaccharide (LPS) (L4391, Sigma-Aldrich) and cultivation was continued for 6 or 18 h. Supernatants were harvested after 6 h by centrifugation at 450 × g and 7000 × g and frozen at −80 °C. MDM phenotype was assessed after 18 h of co-culture by flow cytometry.