Ll 37

Whole cell genomic DNA was isolated from PBMCs of each subject using the QiAmp DNA Mini Kit (Qiagen, Hilden, Germany). Self-DNA was incubated with a recombinant antimicrobial peptide, LL37 (AnaSpec, CA, USA), at different ratios in 1X phosphate buffer saline (PBS) (pH 7.4) at room temperature (RT) at different time points (5–60 minutes) for standardization of optimal formation of DNA-LL37 complexes. Finally, in all experiments, 1 µg of DNA was mixed in 5 µg of LL37 peptide in 20 µl PBS^{42 (link)}, and multiple aliquots were formed for experiments and stored at −20 °C to avoid batch-to-batch variations. Free DNA was removed by DNase 1 (4 units in 2 µL, Sigma-Aldrich, USA) treatment for 30 minutes at RT and the reaction was stopped with 1 M NaCl. The DNA-LL37 complexes were then diluted in complete RPMI medium at a final concentration of 3 µg/100 µL and added to pDC and monocytes in cultures according to experimental requirement. In each experiment, genomic DNA used in the formation of DNA-LL37 complexes and cells (pDCs, monocytes, CD4 + T and CD8 + T cells) used for various assays, were obtained from the same subject.

hMSCs isolated from bone marrow of healthy persons under informed consent were purchased from Lonza (Cologne, Germany). Each hMSC lot used in the study was tested by Lonza for purity and their ability to differentiate into the osteogenic, chondrogenic, and adipogenic lineage. The cells were positive for CD29, CD44, CD105, and CD166, and negative for CD14, CD34, and CD45. hMSCs were cultured as described previously,^{28 (link)} using the StemMACS expansion media with a weekly medium change (Miltenyi Biotec, Bergisch Gladbach, Germany). All studies were carried out with hMSCs between the fifth and seventh passage of cultivation which exhibited an average cell doubling time of 96 h. For experiments under serum-free conditions, hMSCs were washed with serum-free medium and incubated in Dulbecco Modified Eagle Medium (DMEM) (PAA Laboratories, Coelbe, Germany) supplemented with 1% Nutridoma SP (Roche Applied Science, Mannheim, Germany) in the absence or presence of LL-37 (Anaspec, Fremont, CA, USA) at physiological concentrations of 10–100 ng/mL LL-37 as determined in human blood plasma.^{30 (link)} Cells were routinely tested for mycoplasma contamination. Cell viability was determined by trypan blue staining and periodically by application of the WST-8 assay (Dojindo, Rockville, MD, USA).

qRT-PCR was used to determine TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 mRNA levels. Purified mast cells suspended in cDMEM were stimulated with LL-37 (AnaSpec, Fremont, CA, USA) at a final concentration of 1 μg/mL in a humidified atmosphere with 5% CO₂ at 37°C for 1, 3, or 6 h. For control, mast cells were incubated under the same conditions without LL-37. qRT-PCR was conducted as previously described [10 (link), 28 (link)]. The mRNA expression was corrected by normalization based on the transcript level of the housekeeping gene rat Actb.