Study population, study design and recruitment. The ethical committee of the Medical University of Innsbruck approved the study (EC numbers: 1100/2020 and 1111/2020), which took place between April 21 and 27, 2020. This cross-sectional epidemiological survey targeted all residents of Ischgl/Tyrol irrespective of age and gender. At the time of investigation, Ischgl had a population size of 1,867 individuals, 1,617 with their main residence in Ischgl and 250 seasonal immigrant workers, living in 582 different households.
Quantification of immunoproteins. Samples from all groups were measured for the presence and quantity of several human inflammatory cytokines/chemokines, key targets, which are involved in inflammation and immune response and human proteins that are involved in response to viral infections. For this purpose, participants‘ plasma was examined by using four different pre-defined LEGENDplex™ assays (BioLegend, California, USA), while the human inflammation panel 1 was used to quantify 13 human inflammatory cytokines including IL-17A, IL-18, IL-23 and IL-33, the pre-defined human inflammation panel 2 quantified TGF-β1, sTREM1, PTX-3, sCD40L, sCD25, CXCL12, sST2, sTNF-RI, sTNF-RII, sRAGE and CX3CL1. The human anti-virus response panel measured quantities of type 1 interferons (INF-α2, IFN-β), type 2 interferons (IFN-γ), and type 3 interferons (IFN-λ1, IFN-λ2/3) as well as the interleukins IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α, IP-10 and GM-CSF. Additionally, chemokines were analyzed using the human proinflammatory chemokine panel. The LEGENDplex assays were used according to the manufacturer ‘s instructions. In short, immunoprotein capturing by antibody-bearing beads was done by using V-bottom plates. Bead-bound proteins were measured with a FACS CantoII cytometer (Becton Dickinson, New Jersey, USA), whereby fluorescent signal intensity was proportional to the amount of bead-bound proteins. The concentrations of the immunoproteins were calculated with the LEGENDplex™ data analysis software.
Extraction of the buffy coat and purification of RNA. Extraction of the buffy coat and subsequent RNA purification will be performed as described10. In short, the drawn blood is centrifuged at 1,600g for 10 min at 4°C. After vacuming off the plasma layer, the buffy coat layer is carefully collected. The obtained buffy coat is mixed with 1 mL RBC lysis buffer and incubated for 10 min at room temperature. After a centrifugation step, supernatants were discarded, and the pellets mixed with 1 mL RBC lysis buffer. The pellet is washed with PBS buffer and then mixed with 1 mL TRIzol®. For extraction of the RNA the TRIzol reagent single-step method will be used11. After addition of 2 M sodium acetate (pH 4), the tube is mixed thoroughly by invertion before adding chloroform/isoamyl alcohol (49:1) and another mixing step. The sample gets incubated on ice for 15 min and subsequently centrifuged for 20 min at 10,000g and 4°C. The obtained aqueous phase is transferred to a new Eppendorf tube, 1 mL isopropanol is then added, which is followed by an incubation at -20°C for 1 h to precipitate RNA. The final RNA precipitate is won by centrifugation at 10,000g at 4°C and discarding of the supernatant. Pellets are stored at -80°C until dispatch.
mRNA sequencing (mRNA-seq) and data analysis. Nanophotometer (Implen) was used to analyze each sample for concentration and the RNA quality was assessed by an Agilent Bioanalyzer 2100 (Agilent Technologies). The Poly-A containing mRNA is purified by poly-T oligo hybridization from 1 μg of total RNAs and cDNA was synthesized using SuperScript III (Invitrogen). Libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded mRNA Library Prep Kit (Illumina, RS-20020595) and paired-end sequencing was done with a NovaSeq 6000 instrument (Illumina).
mRNA-seq read quality control was done using Trimmomatic24 (version 0.36) and STAR RNA-seq25 (version STAR 2.5.4a) using 150bp paired-end mode was used to align the reads (hg19). HTSeq26 was to retrieve the raw counts and subsequently, R (https://www.R-project.org/), Bioconductor27 and DESeq228 were used. Additionally, the RUVSeq29 package was applied to remove confounding factors. The data were pre-filtered keeping only those genes, which have at least ten reads in total. Genes were categorized as significantly differentially expressed with an adjusted p-value (pAdj) below 0.05 and a fold change > 2 for up-regulated genes and a fold change of < -2 for down-regulated ones. The visualization was done using dplyr (https://CRAN.R-project.org/package=dplyr) and ggplot230. The genes were cutoff in the standard, less than 5 value and less than 1 log2 fold change and then conducted gene enrichment analysis (https://www.gsea-msigdb.org/gsea/msigdb).
Statistical analysis. For comparison of protein levels, single sample pairs were evaluated with the unpaired two-tailed t-test to compare the distributions of two groups with the Welch’s t test to compare the two distributions (GraphPad PRISM version 8.2.0). For comparison of RNA expression levels between CF patient and asymptomatic seropositive cohort, a two-way ANOVA followed by Tukey’s multiple comparisons test was used (GraphPad PRISM). P<0.05 was considered statistically significant.
Data availability. The RNA-seq data for patients will be uploaded in GEO.