Methods

Human Study PreDicta

In the European Study PreDicta (Post-infectious immune reprogramming and its association with persistence and chronicity of respiratory allergic diseases) we examined healthy and asthmatic pre-school children at the age of 4-6 years in collaboration with the children hospital in Erlangen. The study in Erlangen was approved by the ethics committee of the Friedrich-Alexander University Erlangen-Nürnberg, Germany (Re-No 4435) and it is registered in the German Clinical Trials Register (www.germanctr.de: DRKS00004914).
Two cohorts of pre-school children (age 4-6 years) with and without asthma were analyzed. The recruitment of the subjects, inclusion and exclusion criteria as well as the timescale for clinical visits and data collection were exactly described recently (Bergauer et al., 2017b; a; Bielor et al., 2017; Hentschke et al., 2017) along with the clinical aspects and characteristics and reported in other form in Table 1 and 2.
For gene expression analysis we isolated mRNA from total blood cells of the children as previously described and performed quantitative real-time PCR as described below (Bergauer et al., 2017a). The levels of CRP in the serum samples of the children were measured by turbidimetry on a Roche Integra 800 Analyzer (CRPL2 reagent, limit of detection 1.0 mg/L, interday CV 1.4% (8.1 mg/L), Roche Diagnostics, Basel). The detection of rhinovirus in nasopharyngeal swab obtained from the children was performed at the Department of Virology, University of Turku (Finland). The description of this procedure is already published in detail elsewhere (Bergauer et al., 2017a).

FEV1 and PEF

FEV1, FVC and PEF were measured at Baseline visit (B0) by using spirometry. After a period of normal breathing, the participant should inhale maximal, directly followed by maximal and fast exhalation. The volume exhaled in one second is FEV1. The total exhaled volume is FVC. The ratio FEV1/FVC is stated as FEV1%. The PEF is defined as the largest expiratory flow, which is achieved with a maximum forced effort after maximum inspiration.

Human RNA Isolation from Tempus Tubes and quantitative Real-Time PCR

At Baseline visit whole blood was collected in Tempus® Blood RNA Tubes (Life Technologies™, GmbH, Darmstadt, Germany) and RNA was extracted with the MagMax for Stabilized Blood Tubes RNA Isolation Kit. For reverse transcription of RNA (1 µg) we used the first strand cDNA synthesis kit for RT-PCR (MBI Fermentas, Sat. Leon-Rot, Germany). The resulting template cDNA was then amplified by quantitative real-time PCR (qPCR) using SoFast EvaGreen Supermix (Bio-Rad Laboratories, München, Germany). The qPCR itself was performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with a cycle of 2 min 98°C, 50 cycles at 5 s 95°C, 10 s 60°C, followed by 5 s 65°C and 5 s 95°. The primer sequences used for the for real time PCR are listed in Table S1. The mRNA of the genes of interest was normalized using the housekeeping gene HPRT (Hypoxanthine Guanine Phosphoribosyl Transferase).

Isolation of PBMCs, in vitro cell culture and analysis of the cell supernatants

At the time of recruitment (Baseline Visit), PBMCs were isolated from heparinized blood with Ficoll using density centrifugation. After isolation, PBMC numbers were adjusted to a concentration of 106 viable cells/mL in complete culture medium. For cell culture, RPMI 1640 medium supplemented with 25 mmol/L HEPES (GIBCO, Invitrogen, Darmstadt, Germany) was used. Furthermore, 100 IU/mL penicillin, 100 µg/mL streptomycin, 50 µmol/L β-mercaptoethanol, 1% L-glutamine (200 mmol/L), 1% MEM Vitamin, 1% non-essential amino acids, 1% sodium pyruvate, and 10% FBS were added (complete culture medium); these reagents were purchased from Sigma-Aldrich (Steinheim, Germany). The PBMCs were cultured in complete culture medium for 24 hours at 37°C and 5% CO2, whereby parts of them were challenged in vitro with 10 µg/ml PHA (Sigma-Aldrich, Steinheim, Germany) or with RV (RV1b). The growth of RV1b and the description of the RV1b infection itself have been published previously in detail elsewhere (Bergauer et al., 2017a).
Human IFNβ and IL-10 was detected in the cell-culture supernatants by using IFNβ ELISA kit from PeproTech (Hamburg, Germany) and IL-10 OptEIA™ sandwich ELISA kit from BD Bioscience (Heidelberg, Germany), respectively, according to the manufacturer’s protocol.

Statistical analysis

Statistical analysis was performed using Prism version 7 for Windows (GraphPad, La Jolla, CA, USA). Differences were evaluated for significance by using the two-tailed Student’s t test or ordinary One-way ANOVA to generate p-value data (* p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, **** p≤ 0.0001) for all data. Unless otherwise indicated data are presented as mean values ± SEMs.