Baker's yeast beta glucan supplementation was associated with an improved innate immune mRNA expression response after exercise.

Beta glucans are found in many natural sources, however, only Baker's Yeast Beta Glucan (BYBG) has been well documented to have structure-function effects that are associated with improved innate immune response to stressors (e.g., exercise, infection, etc.). The purpose was to identify a BYBG-associated mRNA expression pattern following exercise. Participants gave IRB-approved consent and were randomized to BYBG (Wellmune®; N=9) or Placebo (maltodextrin; N=10) for 6-weeks prior to performing 90 min of whole-body exercise. Paxgene blood samples were collected prior to exercise (PRE), after exercise (POST), two hours after exercise (2H), and four hours after exercise (4H). Total RNA was isolated and analyzed for the expression of 770 innate immune response mRNA (730 mRNA targets; 40 housekeepers/controls; Nanostring nCounter). The raw data were normalized against housekeeping controls and expressed as Log2 fold change from PRE for a given condition. Significance was set at p < 0.05 with adjustments for multiple comparisons and false discovery rate. We identified 47 mRNA whose expression was changed after exercise with BYBG and classified them to four functional pathways: 1) Immune Cell Maturation (8 mRNA), 2) Immune Response and Function (5 mRNA), 3) Pattern Recognition Receptors and DAMP or PAMP Detection (25 mRNA), and 4) Detection and Resolution of Tissue Damage (9 mRNA). The identified mRNA whose expression was altered after exercise with BYBG may represent an innate immune response pattern and supports previous conclusions that BYBG improves immune response to a future sterile inflammation or infection.

Dry blood spot samples to monitor immune-associated mRNA expression in intervention studies: Impact of Baker's yeast beta glucan.

Monitoring immunological response to physical stressors in a field setting is challenging because existing methods require a laboratory visit and traditional blood collection via venipuncture. The purpose of this study was to determine if our optimized dry blood spot (DBS) methodology yields sufficient total RNA to quantify the effect of Baker's Yeast Beta Glucan supplementation (BYBG; Wellmune; 250 mg/d) on post-exercise mRNA expression. Participants had venous DBS samples collected prior to (PRE), and immediately (POST), 2 (2H), and 4 (4H) hrs after completion of a 90 min run/walk trial in a hot, humid environment. Total RNA extracted from DBS was analyzed using a 574-plex Human Immunology mRNA panel (Nanostring). BYBG supplementation was associated with the increased expression of 12 mRNAs (LTB4R, PML, PRFM1, TNFRSF14, LCK, MYD88, STAT3, CCR1, TNFSF10, LILRB3, MME, and STAT6) and decreased expression of 4 mRNAs (MAP4K1, IKBKG, CD5, and IL4R) across all post-exercise time points. In addition to individually changed mRNA targets, we found eleven immune-response pathways that were significantly enriched by BYBG following exercise (TNF Family signaling, immunometabolism, oxidative stress, toll-like receptor (TLR) signaling, Treg differentiation, autophagy, chemokine signaling, complement system, Th2 differentiation, cytokine signaling, and innate immune). The present approach showed that DBS samples can be used to yield useful information about mRNA biomarkers in an intervention study. We have found that BYBG supplementation induces changes at the mRNA level that support the immune system and reduce susceptibility to opportunistic infection (i.e., upper respiratory tract infection) and facilitate improved physical recovery from exercise. Future studies may look to use DBS sampling for testing other nutritional, health, or medical interventions.

Magnetic Isolation, RNA Extraction, and Nanostring Analysis of Human Peripheral Blood Leukocytes Subsets.

Advancements in multiplexed molecular biology techniques have allowed for blood samples and specific circulating blood leukocytes to be a useful sample source when examining systemic changes associated with changes in body weight, muscle injury, disease onset/progression, and other common conditions. One gap in the current scientific knowledge relates to the impact of changes in individual leukocyte subsets on the overall systemic response. While many studies have published data related to changes observed in a mixed population of circulating leukocytes (i.e., whole blood sample), few studies have identified which cell(s) are responsible for the overall change. Since it is established that leukocyte subsets respond differently to various experimental challenges, it may be possible to gain new insight regarding overall biological state. This has application to a variety of health, nutrition, and exercise intervention models. Despite the need to examine changes in mRNA expression levels in individual leukocyte subsets they are not always easy to isolate and perform mRNA analysis on. In this report we describe a method to magnetically isolate, stabilize RNA, and analyze 800+ mRNA in a single sample analysis. Further we compared total leukocyte and leukocyte subset (i.e., granulocytes, monocytes, and T-cells) mRNA expression to better understand how subset changes contribute to overall response. Examination of subset responses may provide targets for future intervention studies. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Automated magnetic isolation of granulocytes, monocytes, and T-cells Basic Protocol 2: RNA extraction from magnetically sorted granulocytes, monocytes, and T-cells Basic Protocol 3: Nanostring analysis of RNA extracted from magnetically sorted granulocytes, monocytes, and T-cells.

Optimization of RNA Extraction from Dry Blood Spots for Nanostring Analysis.

Dry blood spot (DBS) technology has been widely used since the 1960's for the detection of protein biomarkers associated with various disease states. In this manuscript we report a revised approach using DBS samples to extract total RNA for use in downstream multiplex RNA detection methodology (Nanostring). To accomplish this objective, we have used commercially available supplies, kits, and equipment to ensure that the procedure described in this report can be adopted by any laboratory. The methods described in this report allow for the extraction of high-quality, total RNA from a minimal volume (∼200 µl) of DBS spots. The isolated RNA can be analyzed using a multiplex, Nanostring system to yield results for up to 800 RNA targets. Additional bioinformatics and pathway annotation can be conducted to determine changes in biological signaling pathways. © 2023 Wiley Periodicals LLC. Basic Protocol: RNA extraction from DBS for multiplex RNA nanostring analysis Support Protocol 1: RNA extraction from PAXgene blood Support Protocol 2: Concentration of DBS RNA.