Materials
The following reagents were used in this study: HNP1, HNP2 and HNP3 (Peptide Institute, Inc., Japan); TGF- (BioLegend Inc., CA, USA); Dulbeccos Modified Eagles Medium (Cytiva, Marlborough, MA, USA); Fetal Bovine Serum (Gibco, Grand Island, NY); ProLong Gold Antifade Mountant with DAPI (Invitrogen, CA, USA); LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision Inc., CA, USA); RNeasy Mini Kit (QIAGEN Inc., Hilden, Germany); iScript Reverse Transcription Supermix, SsoAdvanced Universal Probes Supermix (Bio-Rad Inc., CA, USA); Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., NY, USA); 1X Protease/Phosphatase Inhibitor Cocktail, Rabbit anti-COL1A1 antibody, Mouse anti-Ki-67 antibody, Rabbit anti--actin antibody, Mouse anti-rabbit IgG antibody (HRP conjugate), Anti-rabbit IgG Alexa Fluor 555, Anti-mouse IgG Alexa Fluor 488 (Cell Signaling Technology Inc., MA, USA); Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Sciences Inc., MA, USA); Alliance Q9 chemiluminescence imaging system (Uvitec Inc., UK); Tissue-Tek O.C.T. Compound (Sakura, Alphenaan den Rijn, Netherlands).
Neonatal foreskin tissues were obtained by surgical circumcision of healthy male neonates at the Pediatric Surgery clinic, King Chulalongkorn Memorial Hospital with parental informed consent and assent forms. Ethical approval for this study was granted by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB 120/63). We confirm that all methods and experiments were performed in accordance with relevant guidelines and regulations. Dermal fibroblasts were isolated as described previously17 and cultured in medium containing DMEM supplemented with 10% FBS and gentamicin (1mL/L). The cells were incubated in a 5% CO2 incubator at 37C, and the cells derived from the 2nd to 5th passage were used in experiments.
Dermal fibroblasts (5103 and 1104 cells/well) in 100 L of DMEM with 10% FBS were seeded into 96-well clear round bottom, ultra-low attachment plates. The medium was replaced with fresh medium every 3days18. Spheroids were imaged at days 3, 5 and 7 and diameters were measured by ImageJ.
Cell proliferation was analyzed by methylene blue staining. Dermal fibroblasts were seeded into a 96-well plate (3103 cells/well) with 1% FBS DMEM overnight. HNP1-3 (0.62510M) were added into the wells, and the cells were incubated for 24h. The supernatant was collected, and the cells were fixed with 20% (v/v) formaldehyde for 48h and stained with methylene blue for 30min. The cells were washed and eluted with 100 L of ice-cold HCl (0.1M) in absolute ethanol solution (1:1 ratio). The absorbance was measured at 650nm using microplate reader. Cytotoxicity was analyzed using LDH-Cytotoxicity Colorimetric Assay Kit II. Collected supernatants (2.5 L) were mixed with 25 L of LDH reaction mix for 30min. Stop solution (2.5 L) was added and the absorbance was measured at 450nm using microplate reader. Spheroids derived from dermal fibroblasts (5103 cells/well) were treated with HNP1-3 at 10M for 4days. All experiments were performed in triplicates.
Dermal fibroblasts were seeded into a 6-well plate (2.5105 cells/well) in DMEM containing 1% FBS overnight. The cells were treated with HNPs (2.5, 5 and 10M) for 24h. Total RNA was extracted and converted to cDNA with the following conditions: 25C for 5min, 46C for 20min and 95C for 1min. COL1A1 and Ki-67 gene expressions were determined by real-time PCR. ABL gene expression was used as internal control. Primers and probes are listed in Supplementary Table S1 online. Real-time PCR was performed for 40 cycles with the following program: 95C for 2min, 95C for 5s and 60C for 30s.
Dermal fibroblasts were seeded into a 6-well plate (2.5105 cells/well) in 1% FBS in DMEM overnight. HNPs (2.5, 5 and 10M) were added into the wells and the cells were incubated for 48h. Cells were lysed by 1X RIPA Lysis Buffer containing 1X Protease/Phosphatase Inhibitor Cocktail. Total protein concentration was measured by Pierce BCA Protein Assay Kit. Protein lysates (10g) were mixed with 2X SDS dye and heated at 100C for 5min. Proteins were loaded in 7.5% SDS-PAGE and gel electrophoresis was performed at 100V for 1.5h. Proteins were transferred to PVDF membrane with electrophoresis at 15V for 50min. Blotting membranes were blocked with 1X PBS with 0.1% Tween-20 (PBST) containing 5% skimmed milk, followed by incubation with primary antibodies; COL1A1 (1:2000) and -actin (1:4000), overnight at 4C. The membranes were washed with PBST, and mouse anti-rabbit IgG (HRP conjugate) secondary antibody (1:4000) was added. The membranes were incubated for 1h with shaking before washing. The membranes were soaked in chemiluminescent substrate (Amersham ECL Western Blotting Detection Kit) and chemiluminescence signals were directly scanned with Alliance Q9 chemiluminescence imaging system. The band intensity was quantified by densitometry using ImageJ.
Dermal fibroblasts were seeded into a Lab-Tek II Chamber Slide System (1.5104 cells/well) in 1% FBS in DMEM overnight. HNPs (2.5, 5 and 10M) were added into the cells and incubated for 24h. The cells were washed with PBS and fixed with 4% paraformaldehyde for 10min. The cells were treated with 0.2% Triton-100 in PBS for 2min and blocked with 1% BSA in PBS for 30min. Primary antibody: Ki-67 (1:1000), diluted in 1% BSA in PBS was added and the cells were incubated at 4C overnight. After washing, secondary antibody: anti-mouse IgG Alexa Fluor 488 (1:2000), diluted in 1% BSA in PBS was added and the cells were incubated for 1h. After washing, the sections were mounted and proteins were observed.
Spheroids derived from dermal fibroblasts (5103 cells/well) were treated with HNPs (10M) for 4days. The spheroids were collected and covered with Tissue-Tek O.C.T. Compound. Frozen spheroids were cryosectioned into 8m thick layers onto glass slides. The sections were washed with PBS, fixed with 4% paraformaldehyde for 10min and treated with 0.2% Triton-100 in PBS for 2min. The sections were blocked with 5% BSA in PBS for 1h and incubated with primary antibody: COL1A1 (1:400), diluted in 1% BSA in PBS at 4C overnight. After washing, the sections were incubated with secondary antibody: anti-rabbit IgG Alexa Fluor 555 (1:1000), diluted in 1% BSA in PBS for 1h. After washing, the sections were mounted and proteins were observed.
The statistical analyses were determined by paired t-test using GraphPad Prism 9.0.0 (GraphPad Software, Boston, MA, USA). A simple linear regression analyses was performed using STATA version 15.1 (StataCorp, College Station, TX USA). The regression coefficients, 95% confidence intervals (CI), and p-value were demonstrated. The results were expressed as the meanstandard deviation (SD) and differences with a p-value<0.05 were considered statistically significant.
Dermal fibroblasts were seeded into a 6-well plate (2.5105 cells/well) in DMEM containing 1% FBS overnight. The cells were treated with HNP1 (10M) for 24h. Total RNA was extracted and the quality of extracted RNA (RNA Integrity Number6.5) was evaluated using an Agilent 2100 Bioanalyzer. The RNA-seq experiment was conducted by Vishuo Biomedical, Thailand. Purified poly-A mRNA was fragmented, and pair-end RNA sequencing was performed on the Illumina HiSeq platform. The Gene Expression Omnibus (GEO) of raw reads in FASTQ files was GEO ID: GSE230670.
Quality of raw reads in FASTQ files was inspected with the FASTQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The Trim Galore program (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to cut adaptors and sequence reads with a Phred score lower than 30. To estimate abundance of transcript, cleaned raw reads were analyzed with Salmon v1.9.019 by 2 steps; (1) indexing and (2) quantification. First, Salmon with default setting was used to build an index on human reference transcriptome (GRCh38) downloaded from Human Genome Resource at NCBI (downloaded; July 2022) (https://www.ncbi.nlm.nih.gov/projects/genome/guide/human/). Next, Salmon was used for quantification by mapping paired-end reads to the indexed reference sequence in mapping-based mode. Transcript abundances in estimated read counts were imported to R with tximeta v1.12.420 and aggregated to gene-level expression with gene model annotation (GRCh38) for further analysis. Principal component analysis (PCA) was performed on the pre-processed gene expression data, which were first log-transformed and normalized with respect to library sizes by the rlog function in DESeq221 package and standardized so that the expression level of each gene has a zero mean and a unit variance, to visualize the clustering structure of replicates. PCA plots were drawn in R using the ggplot2 package.
Differential gene expression was tested between HNP1 and control groups with DESeq2 v1.34.021 package. Gene expression was normalized with the median of ratios method from DESeq2. Since samples were derived from different donors, statistical design for DESeq2 was accounted for donor factor when fitted generalize linear model to data. Multiple hypothesis testing correction was performed using Benjamini-Hochberg's procedure. Differentially expressed genes (DEGs) were defined as genes with false discovery rates (FDR)<0.01. Boxplots were drawn in R using ggplot2.
Function of genes was analyzed with gene set enrichment analysis (GSEA) from WebGestalt (http://www.webgestalt.org/)22. The values of log fold changes were used to rank genes for the functional enrichment analysis using Gene Set Enrichment Analysis (GSEA) method. KEGG pathway and Gene Ontology databases (biological process, molecular function and cellular component) were used. Multiple hypothesis testing correction was performed using Benjamini-Hochbergs procedure with the FDR cutoff of 0.05 for enriched functions.
Link:
Regulation of dermal fibroblasts by human neutrophil peptides ... - Nature.com
