## 4 Materials and Methods
### PBMC Stimulation Assays  
For all assays in PBMCs, cells were carefully thawed and incubated in supplemented RPMI and cell count and viability was measured on Countess Automated Cell Counter (Life Technologies, Grand Island, NY, USA). Cells were seeded in 96-well plates, 0.5 million cells per well in every assay and incubated at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\). All supernatants were stored at \(-\)80 \({}^{\circ}\)C. Unstimulated cells served as controls in all assays. As mechanisms for CCL20 release from PBMCs have not been thoroughly investigated, we performed a pilot study to select putative ligands. PBMC from four healthy individuals were stimulated with ligands to toll-like receptor (TLR) 2/1 (pam3 cysSK4-P3C), TLR2/6 (lipomannan-LM), TLR3 (Poly IC), TLR4 (lipopolysaccharide-LPS), TLR5 (flagellin-Flag), TLR7/8 (R848), TLR9 (CpG), NOD2 (muramyl dipeptide-MDP), proinflammatory cytokines interleukin 1 beta (IL-1\(\beta\)) and tumour necrosis factor (TNF) and the anti-inflammatory cytokine interleukin 10 (IL-10). Supernatant was collected after six and 20 h of stimulation. Following the pilot study, a stimulation assay was conducted in PBMCs from 40 individuals. Ligands yielding less than 200 pg/mL CCL20 in the pilot study were excluded (except for IL-10 as it is anti-inflammatory). PRR ligands and cytokines used for stimulation were MDP and Flagellin (both at 100 ng/mL) from Invivogen (San Diego, CA, USA) P3C (300 ng/mL) from EMC (Tuebingen, Germany), CpG (10 \(\upmu\)M) from TibMolBiol (Berlin, Germany), IL-1\(\beta\), IL-10 and TNF, all at 100 ng/mL from PeproTech (Rocky Hill, NJ, USA). Supernatant was collected after 20 h of stimulation. To determine whether CCL20 release from PBMCs occurred mainly secondarily to IL-1\(\beta\), IL-1\(\beta\) neutralization assays were performed in PBMCs from three healthy donors. Cells were treated with anti-hIL-1\(\beta\)-IgG (cat.nr mabg-hillb-3) or Mouse IgG1 isotype control (cat.nr mabg1-ctrlm) from Invivogen (San Diego, CA, USA) (1 \(\upmu\)g/mL) for one hour prior to stimulation for six h with P3C (300 ng/mL), MDP (100 ng/mL) and IL-1\(\beta\) (100 ng/mL). Further, we performed CCL20 stimulation in PBMCs to investigate if CCL20 has additional functions to chemotaxis and antibacterial traits. In a pilot experiment, PBMCs from four healthy individuals were stimulated with CCL20 alone or in combination with TNF. Multiplex analysis covering 27 cytokines (Bio-Plex Pro, Human cytokine 27-plex Assay, Bio-Rad, Hercules, CA, USA) was used to screen for effects. Based on the pilot assay, PBMCs from 40 subjects were stimulated for 20 h with CCL20 (100 ng/mL) (R&D systems, Abington, UK) alone or in combination with TNF (100 ng/mL). To assess interindividual variability, we conducted the same CCL20 stimulation assay on PBMCs collected from three healthy controls. PBMCs from three healthy individuals were isolated from buffy coat and stored in multiple aliquots on liquid nitrogen. Three separate stimulation assays were conducted on frozen aliquots of PBMCs from all three donors. PBMCs from each individual were thawed and seeded with supplemented RPMI and stimulated following the same protocol as described above.