RA Patients recruitment and PBMCs isolation
Participants at least 18 years old with RA (n=20; mean age=61.5 years old) were recruited for assessing the intracellular levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β and IL-8) expressed by purified monocytes after LPS-stimulation with or without PNLA. While for the transcriptome study, 8 ACPA positive RA patients with active disease (DAS28 score range: 4.8-5.7); (mean age=57 years old) and they were on biologic anti rheumatic drugs. All patients were recruited from the Rheumatology Department at the University Hospital of Wales, signed informed consent was obtained from all participants. The study was approved by the Research Ethics Committee for Wales 3 (reference no. 12/WA/0045). Detailed demographic and laboratory data are described in (Supplementary Tables S1, S2) for intracellular cytokines and WGT assessment, respectively. PBMCs were isolated by standard Ficoll density gradient centrifugation and methodological details are provided in (Supplementary Data S1).
Negative selection and sorting of purified monocytes
To enrich purified monocytes for assessment of intracellular cytokines using flow cytometry, monocytes were recovered by negative selection in cascades by magnetic-activated cell sorting (MACS) pan monocyte isolation kit (Miltenyi Biotec, UK) following the manufacturer’s protocol. Pan monocyte isolation kit (Miltenyi Biotec, UK) uses a cocktail of biotinylated antibodies to select non-CD14+ cells, these cells are magnetically labelled using anti-biotin microbeads and separated from target CD14+ monocytes cells using a magnetic column. 1x107 PBMCs were re-suspended in 100 µl MACS (0.5% BSA, 5 mM EDTA, and 0.09% sodium azide in PBS), and 10 µl of FcR blocking reagent and 10 µl of biotin-antibody cocktail (provided in the kit) were added. The suspension was then mixed and incubated for 10 min at 2-8°C. A 20 µl MACS buffer and 20 µl of anti-biotin microbeads were added /1x107 PBMCs. This suspension was mixed gently and incubated for another 10 min at 2-8°C before passing through a sterile magnetic column. The column was then washed with MACS buffer to ensure all purified monocytes were collected in a sterile 15 ml Falcon tube. The flow through purified monocytes were centrifuged at 350 x g for 5 min at room temperature. Number of viable cells were determined via trypan blue exclusion staining and counted using a haemocytometer and light microscope. Cells at a minimum density of 106 cells/ml were cultured in complete RPMI medium at 37°C in 5% CO2 controlled environment; the procedure was continued for cell surface/intracellular staining as described below or for the transcriptome analysis.
For the transcriptome study, the magnetically purified monocytes were passed through a second step of purification using FACS Aria III based cell sorting. In brief, cells were prepared for sorting, enriched monocyte cell suspensions were incubated with 2 μg/ml Fc human serum immunoglobulin (BD Biosciences; UK) at 4°C for 10 min to reduce non-specific binding by Fc receptors, and a small volume was aliquoted and labelled as unstained that was used for compensation. The cells were then stained with live/dead™ (L/D) fixable viability stain (ThermoFisher Scientific, UK) with fluorochrome-conjugated antibodies to CD14 (BioLegend, USA) at a final dilution of 1/100. All experiments were controlled with appropriate isotype antibodies, compensation beads and unstained cells. To verify the purity and quantity of the sorted monocytes, purity check was performed under sterile conditions at 0-4°C. The purity of the monocytes was 83.3%, 83%, 92.3%, 85.2%, 89.2%, 87%, 90.7%, and 96% respectively for patients RA1- RA8. Representative flow cytometry plots of the sorted monocytes population used in the study are shown in Supplementary Fig. S1.
Monocyte treatment, cell surface and intracellular cytokines staining
For assessment of intracellular cytokines by flow cytometry, enriched monocytes were seeded in 12-well plates at a minimum density of 106 cells/well in 1 ml of RPMI 1640 complete medium (RPMI 1640 medium containing 10% foetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine) along with 25 and 50 µM PNLA (Cayman Chemicals, USA) or DMSO (Sigma-Aldrich, UK) vehicle control for 24 hour and some wells were stimulated with LPS (100 ng/ml) and Brefeldin (PFA; 10 µg/ml) both (Sigma-Aldrich, UK) for 8-9 hour or left un-stimulated as a control and the culture continued. Following all incubations, the monocytes were collected by detachment of the plastic wells (Supplementary data S2) and stained for surface fluorochrome-conjugated antibodies specific to monocyte surface receptors CD14, and CD16 (BioLegend, USA) as described in Supplementary data S2. The monocytes were then permeabilised using 0.3% saponin (Thermo Fisher Scientific, UK) diluted in PBS and stained for intracellular cytokines panel TNF-α, IL-1β (BioLegend, USA) and IL-6, IL-8 (BD Biosciences, UK) as in Supplementary data S2.
Flow cytometry analysis
A minimum of 106 cells/mL live cells/sample were collected using forward scatter (FSC) and side scatter (SSC) gating to avoid dead cells and debris and at least 50,000 events were acquired using flow cytometer (BD LSR-FORTESSA) in the 2-3-6-5 configuration (16 colour). Details on acquisition and analysis are provided in Supplementary data S2.
Transcriptome analysis
Sorted monocytes were washed with fresh RPMI medium, counted, and seeded in 6-well plates at a concentration of approximately 1x106 cells/mL in RPMI 1640 complete medium along with either 25 mM PNLA or DMSO vehicle control for 24 hours before stimulation with LPS (100 ng/ml) or vehicle control for 4 hours. The cell suspensions were then collected and washed with Ca2+ or Mg2+ free Dulbecco's PBS medium (Thermo Fisher Scientific, UK). The adherent cells were detached from the plastic by addition of 1 ml accuatase cell dissociation reagent (Thermo Fisher Scientific, UK) pH 6.8/well, incubated for 5-8 min at 37°C with 5% CO2, and then washed. The cell suspension was centrifuged at 400 x g at room temperature for 5 min and kept at -80°C in 350 ml buffer RLT (provided in RNeasy Mini kit) supplemented with 10% of 2-mercaptoethanol (Gibco Life Science, UK) as a lysate till RNA extraction.
RNA extraction
Total RNA was isolated using a RNeasy mini kit (Qiagen, Hilden, Germany) from purified monocytes following 100 ng/ml LPS and 25 µM PNLA or vehicle treatment. RNA was purified using a RNeasy kit on-column with DNase I digestion (Qiagen) as described in Supplementary data S3. The cell lysates were stored at −80°C and then passed through a series of spin columns to first bind genomic DNA, then RNA and finally to elute high-quality RNA. A quality control check for RNA was assessed as described in Supplementary data S3.
Library construction, sequencing, and data processing
Library preparation including ribosomal RNA depletion
In collaboration with the Wales Gene Park (Cardiff University), 5 ng of total RNA was depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), (New England BioLabs, (UK) Ltd). Ribosomal RNA depletion of each sample was assessed using the Agilent 4200 TapeStation with hsRNA ScreenTape (Agilent Technologies, Inc, UK).
The sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs, (UK) Ltd) protocol. The steps included RNA fragmentation and priming, 1st strand cDNA synthesis, 2nd strand cDNA synthesis, adenylation of 3’ ends, adapter ligation (adapter diluted 1:199) and PCR amplification (16-cycles). Following the addition of the PCR enrichment master mix, a unique index primer was added to each sample. The standard fragmentation procedure of 15 min at 94°C for samples for intact RNA (>7) was reduced to 8 min at 94°C for partially degraded RNA with RIN values 2-6. Except for the replacement of RNAClean® XP beads and SPRI select beads by AMPure XP beads (Beckman Coulter®) the manufacturer’s instructions were followed. The libraries were validated using Agilent 4200 TapeStation and high sensitivity DNA ScreenTapes (Agilent Technologies, Inc, UK) to ascertain the insert size, and the Qubit® (Thermo Fisher Technologies, UK) was used to perform the fluorometric quantitation.
RNA-sequencing (RNA-seq)
Following library quantification and validation, the normalized library pool was sequenced on one lane of an S1 (200 cycle) flow cell using XP workflow and 2x100 bp paired end dual indexed format on the NovaSeq6000 sequencing system (Illumina® Inc, USA) according to the manufacturer’s instructions. To conform to ENCODE guidelines, libraries were sequenced to have >54 million mapped reads (encodeproject.org/documents/cede0cbe-d324-4ce7-ace4-f0c3eddf5972).
RNA-seq data processing and read mapping strategy
Paired end reads from the Illumina sequencing were trimmed with Trim Galore (7) and assessed for quality using FastQC (8), using default parameters. Reads were mapped to the human GRCh38 reference genome using STAR software (9) and reads per gene were assigned using the featureCounts software (10) with the GRCh38.96 gene build gene transfer format (GTF). Both the reference genome and GTF were downloaded from the Ensembl FTP site (11). Duplicate reads were identified and marked using MarkDuplicates in Picard (Broad Institute).
Normalisation and identification of differentially expressed genes (DEGs)
Differential gene expression analyses used the DESeq2 package (12). Genes were discarded from the analysis if differential expression failed to be significant (p value < 0.05) (12). The initial WGT data were assessed with the web-based tool Morpheus (https://software.broadinstitute.org/morpheus/) using heatmaps to view changes in gene expression. The data were then separated into 3 groups; vehicle treated, vehicle treated/LPS stimulated, and PNLA treated/LPS stimulated monocytes for functional analysis including gene set enrichment analysis, gene ontology and further downstream pathway analysis.
Heatmap of DEGs and principal component analysis (PCA)
A heatmap and PCA are shown in (Supplementary Figs S2, S3). The heatmap was generated using broad Morpheus software and visualizations used fragments per kilobase per million mapped fragments (FPKM) reads and log2 fold change (log2 FC) comparing PNLA-treated and LPS-stimulated monocytes with those treated with vehicle and LPS from RA patients at equivalent time point (Supplementary Figs S2). Data sets were hierarchically clustered using 1 minus Pearson’s correlation coefficient. PCA was performed in R using normalized data from the DESeq2 analysis, and data were clustered using the top 50 most DEGs. (Supplementary Fig S3). Each plot shows the results of the first two principal components.
Statistical analysis
The statistics used were dependant on the experiments performed and depending on data distribution. Normality of data was tested using the Shapiro–Wilk test and confirmed with histograms and Q-Q plots in SPSS Statistics (version 23; IBM Corp., Armonk, NY, USA). FCS data files were analysed using FlowJo (version10.7, BD, UK) and the NoVo express. Percentage of cytokine expressing CD14+ monocytes was calculated using Microsoft Excel (version 16.52, Microsoft 365 Subscription, UK). Graphs and statistical analysis were performed using Graph Pad Prism (version 8 GraphPad Software, La Jolla, CA, USA). One way ANOVA was used to compare multiple groups followed by Dunn’s post hoc analysis for pairwise comparisons. Spearman correlation was used to analyse the linear association using SPSS statistics (version 26; IBM Corp, Armonk, NY, USA) and 2 tailed Mann Whitney U test was applied to find the comparison between 2 groups. All values are presented as mean ± SEM, P- value <0.05 was considered significant for all the analysis.