Digital Immune Gene Expression Profiling Discriminates Allergic Rhinitis Responders from Non-Responders to Probiotic Supplementation

Probiotic supplementation for eight weeks with a multi-strain probiotic by individuals with allergic rhinitis (AR) reduced overall symptom severity, the frequency of medication use and improved quality of life. The purported mechanism of action is modulation of the immune system. This analysis examined changes in systemic and mucosal immune gene expression in a subgroup of individuals, classified as either responders or non-responders based on improvement of AR symptoms in response to the probiotic supplement. Based on established criteria of a beneficial change in the mini-rhinoconjunctivitis quality of life questionnaire (mRQLQ), individuals with AR were classified as either responders or non-responders. Systemic and mucosal immune gene expression was assessed using nCounter PanCancer Immune Profiling (Nanostring Technologies, Seattle, WA, USA) kit on blood samples and a nasal lysate. There were 414 immune genes in the blood and 312 immune genes in the mucosal samples expressed above the limit of detection. Unsupervised hierarchical clustering of immune genes separated responders from non-responders in blood and mucosal samples at baseline and after supplementation, with key T-cell immune genes differentially expressed between the groups. Striking differences in biological processes and pathways were evident in nasal mucosa but not blood in responders compared to non-responders. These findings support the use of network approaches to understand probiotic-induced changes to the immune system.

Nasal lavage samples were collected from participants with a modified nasal lavage procedure. Traditionally nasal lavage samples are collected by tilting the participants head backwards and instilling 5 ml of saline solution into each nasal cavity with fluid recovery after a predetermined dwell time. The method used in the current study was designed to sample a greater surface area of the nasal mucosa and sinuses. Using a nasal irrigation bottle (FLO Sinus Care, ENT technologies, Melbourne, Australia), participants administered 100 ml of PBS into each nostril and collected the fluid that was expelled from the free nostril into a sterile container. A volume of 20 ml of RMPI was then added to the lavage fluid to support the live cells until processing.
Nasal brushing was performed directly after the nasal lavage. Participants were advised to insert a brush (Piksters size 5, Erkstine oral care, Macksville, Australia) between the nasal septum and inferior turbinate and gently rotate the brush in circular and linear movements for 30 secs. The brush was then removed from the nasal cavity and suspended in a sterile container containing 3.5 ml RPMI. The procedure was then repeated in the other nostril with a fresh brush and placed into the same container following brushing. The nasal lavage and nasal brushing solutions were stored on ice until arrival at the laboratory.

Processing of nasal lavage and nasal brushing samples
The nasal lavage fluid was transferred into nuclease-free centrifuge tubes and centrifuged at 300 xg for 20 mins at 4 °C to concentrate cellular material. If substantial mucous was present, the lavage fluid was passed through a 100 µ M filter (Corning, New York, United States) prior to centrifugation. Once centrifuged, the supernatant was removed and the resulting cell pellets resuspended in the residual volume and transferred into 1.5 ml nuclease-free microfuge tubes. In parallel, the nasal brushes were gently shaken in the RPMI solution to dislodge the nasal cells from the brush and discarded. The resulting cell brushing material was transferred into 1.5 ml nuclease free microfuge tubes. The microfuge tubes were spun at 14, 000 xg for 5 mins. The supernatant was removed and the cell pellets resuspended in residual volume. The resuspended cell pellets from the lavage and brushing samples were then pooled into a single 1.5 ml nuclease-free tube and centrifuged again at 14, 000 xg for 5 mins. The entire supernatant was removed and the resulting cell pellet was resuspended in commercially available lysis buffer (RLT, Qiagen, Hilden, Germany). The lysate was stored at -80 °C for downstream processing.

NanoString gene expression analysis
Immune gene expression analysis was undertaken using the NanoString nCounter analysis system (NanoString Technologies, Seattle, WA) using the commercially available nCounter PanCancer Immune Profiling panel kit together with the nCounter panel plus probe set of 30 immune genes (listed in Supplementary Table 5). The PanCancer Immune profiling panel contains n = 730 genes of key inflammatory pathways and n = 40 reference/housekeeping genes. The nCounter system directly detects and counts single-stranded nucleic acid via reporter probes affixed with flurophore barcodes and biotinylated capture-probes attached to microscopic beads. These are then affixed to lanes in cartridge and read in a digital scanner. Following the manufacturers protocol, 100 ng of total RNA extracted from whole blood or 5 µ l of nasal cell lysate was hybridised with probes at 65 °C for 23 hours before being inserted into NanoString Prep Station were the target-probe complex was immobilised onto the analysis cartridge. Cartridges were read by the nCounter Digital Analyser for digital counting of molecular barcodes corresponding to each target at 280 fields of view.
Supplementary Table S1: DEG in blood at baseline between responders and non-responders. There were 414 genes at baseline in blood expressed above background threshold. The table is order by fold change.