Nucleic Acid-Induced Chemokine Expression in Keratinocytes

Dataset Description:

This dataset contains gene expression data from a study investigating chemokine regulation in human keratinocytes following nucleic acid-induced inflammation. The research examined how synthetic RNA and DNA analogs affect chemokine expression patterns, with focus on identifying pattern recognition receptors and signaling pathways involved in the inflammatory response.

Study Overview: Normal human epidermal keratinocytes (NHEKs, third passage) were treated with synthetic nucleic acids poly(dA:dT) and poly(I:C) to mimic self-derived nucleic acid inflammation characteristic of chronic skin diseases. The study demonstrates that cytoplasmic nucleic acids potently induce CCL2, CXCL10, and CX3CL1 expression primarily through NF-κB signaling pathways.

Data Processing: All gene expression data were generated using quantitative PCR (qPCR) with the ΔΔCt method. Expression values are presented as fold-change relative to untreated control samples, with individual donor values listed for each experimental condition. RNA extraction, cDNA synthesis, and qPCR protocols followed standard procedures with appropriate quality controls.

Dataset Applications: This data supports findings published partially in Kelemen et al, 2022, DOI: 10.3390/ijms23010540 (qPCR array) and provides researchers with comprehensive expression profiles for studying chemokine regulation, innate immune signaling in keratinocytes, and inflammatory pathways relevant to skin diseases including psoriasis, atopic dermatitis, vitiligo, and alopecia areata.

Technical Notes: Data are provided as relative expression values normalized to housekeeping genes (GAPDH for most experiments; multiple controls for qPCR array). Each sheet represents independent experimental designs as detailed in the methodology descriptions.

The following sheets are included in the data file:

qPCR_array:

Results of a custom qPCR array performed on normal human keratinocytes (NHEKs), as described in Kelemen et al, 2022 (DOI: 10.3390/ijms23010540). Briefly, third-passage NHEKs were seeded into 6-well plates at a density of 200 000 cells/ml in complete medium. After 24 hours, cells were transfected with 1 μg/mL polydeoxyadenylic acid-polydeoxythymidylic acid double-stranded homopolymer (poly(dA:dT)) (InvivoGene, San Diego, CA, USA) or with 0.666 μg/mL polyinosinic-polycytidylic acid (poly(I:C)) (Sigma Aldrich, Saint Louis, MO, USA) using the X-tremeGene 9 transfection reagent (Roche Diagnostics). Samples were harvested 6 hours after transfection in TRI Reagent ®. RNA isolation was carried out using the Direct-Zol RNA miniprep kit (Zymo Research), genomic DNA contamination was removed using the Turbo DNA-free Kit (Invitrogen, Life Technologies). cDNA was synthesised using the RT2 first-strand cDNA synthesis kit (Quiagen). An RT2 Profiler PCR array (Quiagen) was performed using the Luminaris SYBR-Green mix (Life Technologies). Relative expression was determined by the ΔΔCt method, where the following genes were used as controls: ACTB, B2M, GAPDH, HPRT1,RPLP0, and expression was compared to untreated (control) samples. Relative expression values of each donorare listed in the table.

6hours:

Results of experiments, where third-passage NHEKs were seeded into 6-well plates at a density of 200 000 cells/ml in complete medium. After 24 hours, cells were transfected with 1 μg/mL polydeoxyadenylic acid-polydeoxythymidylic acid double-stranded homopolymer (poly(dA:dT)) (InvivoGene, San Diego, CA, USA) or with 0.666 μg/mL polyinosinic-polycytidylic acid (poly(I:C)) (Sigma Aldrich, Saint Louis, MO, USA) using the X-tremeGene 9 transfection reagent (Roche Diagnostics). Samples were harvested in TRI Reagent® (Zymo Research Corporation, Irvine, CA) after 6 hours and total RNA was isolated according to the manufacturer’s instructions. The potential contamination of genomic DNA was removed using the Turbo DNA-free Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed into cDNA by the Ultrascript 2.0 cDNA Synthesis Kit (PCR Biosystems Ltd., London, UK). Relative expression was determined by the ΔΔCt method, where GAPDH expression was used as control and expression was compared to untreated (control) samples. Relative expression values os each donor are listed in the table.

cytokines:

Third-passage NHEKs were seeded into 6-well plates at a density of 200 000 cells/ml in complete medium. After 24 hours, cells were transfected with 1 μg/mL polydeoxyadenylic acid-polydeoxythymidylic acid double-stranded homopolymer (poly(dA:dT)) (InvivoGene, San Diego, CA, USA) or with 0.666 μg/mL polyinosinic-polycytidylic acid (poly(I:C)) (Sigma Aldrich, Saint Louis, MO, USA) using the X-tremeGene 9 transfection reagent (Roche Diagnostics) or treated with 100 μg/mL IL-17A, 100 μg/mL IL-23, 100 μg/mL IL-12, 50 ng/mL TNFα, 1,33 μg/mL IMQ, 40nM PMA, 500ng/ml LPS or live Cutibacterium. acnes (C. acnes) strain 889 using at a multiplicity of infection (MOI) of 1:100. Cells were harvested at 24 hours post treatment in TRI Reagent® (Zymo Research Corporation, Irvine, CA) at the indicated time points and total RNA was isolated according to the manufacturer’s instructions. The potential contamination of genomic DNA was removed using the Turbo DNA-free Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed into cDNA by the Ultrascript 2.0 cDNA Synthesis Kit (PCR Biosystems Ltd., London, UK).Relative expression was determined by the ΔΔCt method, where GAPDH expression was used as control and expression was compared to untreated (control) samples. Relative expression values of each donor are listed in the table.

silencing:

Third-passage NHEKs were seeded into 6-well plates at a density of 200 000 cells/ml in complete medium. After 24 hours nucleic acid sensing receptor silencing was carried out using siRNA-mediated gene silencing with the ON-TARGETplus SMARTpool for RIG-I, IFIH1, TLR3, cGAS, ZBP1, IFI16, NLRP1, NLRP3, AIM2 siRNAs, or as control the ON-TARGETplus nontargeting pool (Dharmacon, Lafayette, CO, USA) at a final concentration of 40 nM. Transfection was carried out using the X-tremeGENE siRNA transfection reagent (Roche Diagnostics), according to the manufacturer’s instructions. 24 hours after siRNA silencing cells were transfected by 1 μg/mL poly(dA:dT) or 0.666 μg/mL poly(I:C) using the X-tremeGene 9 transfection reagent (Roche Diagnostics). Samples were harvested in TRI Reagent® (Zymo Research Corporation, Irvine, CA) after 6 hours and total RNA was isolated according to the manufacturer’s instructions. The potential contamination of genomic DNA was removed using the Turbo DNA-free Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed into cDNA by the Ultrascript 2.0 cDNA Synthesis Kit (PCR Biosystems Ltd., London, UK). Relative expression was determined by the ΔΔCt method, where GAPDH expression was used as control and expression was compared to untreated (control) samples. Relative expression values os each donor are listed in the table.

inhibitors:

Third-passage NHEKs were seeded into 6-well plates at a density of 200 000 cells/ml in complete medium. 24 hours after seeding cells were incubated for 60 minutes prior to poly(dA:dT)/poly(I:C) transfection with inhibitors for NF-κB (Bay 11-7085, 10 μM; MedChem Express, Monmouth Junction, NJ, USA), STING (H151, 200ng/ml, InvivoGene) STAT-1 (Fludarabine, 10 μM; Sigma Aldrich), STAT-3 (Stattic, 5 μM; Sigma Aldrich), MEK1 (PD98059, 20 μM; Sigma Aldrich), JNK (SP600125, 10 μM; Tocris Bioscience, Bristol, UK) and p38 (SB203580, 10 μM; Tocris Bioscience). Samples were harvested in TRI Reagent® (Zymo Research Corporation, Irvine, CA) 6 hours after poly(dA:dT)/poly(I:C) transfection and total RNA was isolated according to the manufacturer’s instructions. The potential contamination of genomic DNA was removed using the Turbo DNA-free Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed into cDNA by the Ultrascript 2.0 cDNA Synthesis Kit (PCR Biosystems Ltd., London, UK). Relative expression was determined by the ΔΔCt method, where GAPDH expression was used as control and expression was compared to untreated (control) samples. Relative expression values os each donor are listed in the table.

time_course:

Third-passage NHEKs were seeded into 6-well plates at a density of 200 000 cells/ml in complete medium. After 24 hours, cells were transfected with 1 μg/mL polydeoxyadenylic acid-polydeoxythymidylic acid double-stranded homopolymer (poly(dA:dT)) (InvivoGene, San Diego, CA, USA) or with 0.666 μg/mL polyinosinic-polycytidylic acid (poly(I:C)) (Sigma Aldrich, Saint Louis, MO, USA) using the X-tremeGene 9 transfection reagent (Roche Diagnostics). Cells were harvested at 0, 6, 12, 24 and 48 hours post transfection in TRI Reagent® (Zymo Research Corporation, Irvine, CA) and total RNA was isolated according to the manufacturer’s instructions. The potential contamination of genomic DNA was removed using the Turbo DNA-free Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed into cDNA by the Ultrascript 2.0 cDNA Synthesis Kit (PCR Biosystems Ltd., London, UK).Relative expression was determined by the ΔΔCt method, where GAPDH expression was used as control and expression was compared to untreated (0 hours control) samples. Relative expression values of each donor are listed in the table.