Protecting immunomodulation in healthy adults: effects of beer intake on the inflammasome. acronym: pro-beimm

Background and motivation
Epidemiological and observational studies relate low to moderate consumption of fermented alcoholic beverages to health benefits and suggest that they confer better cardiovascular protection than spirits due to their heterogeneous content of non-alcoholic components.

Inflammation is now recognized as a huge burden for public health. Low-grade chronic inflammation occurs in several stages of non-transmissible chronic diseases and is a major risk factor for unhealthy aging in humans. In this regard, moderate alcohol drinking is associated to prevention against diseases involving inflammation and immunity. Conversely, at high drinking levels appears an increase in the levels of inflammatory mediators and the susceptibility to infections, which tend to offset the benefits in terms of health. Unfortunately, this area remains poorly understood.

Based on a prospective intervention study, we have shown the value of moderate and regular beer intake in improving functionality of high-density lipoproteins (HDL) (Padro et al, 2018), which are also known to have anti-inflammatory effects. Up to now, however, a better understanding is needed on the effects of alcohol consumption and type of fermented beverage on molecules of the “inflammasome” cascade, cell sensors for innate immunity and triggering of inflammation.

Objective
The study is designed to investigate the impact of moderate beer intake on the response of the inflammasome pathway in healthy individuals. Specifically, the study intends to determine whether a chronic moderate intake of regular- and alcohol- free beer regulates inflammasome components including the Toll-like-receptor (TLR)-pathway in peripheral blood leukocytes.

Methodology
We will perform transcriptomic studies for coding and non-coding RNA (miRNAs) in blood inflammatory cells and liquid biopsies obtained from adult subjects (N=37), who have been submitted to two sequential intervention periods with traditional and alcohol- free beer (330mL women, 660 mL men/day) as part of a prospective, randomized, single-center human intervention trial with a crossover design (Padro et al, 2018). The study will include (1) High throughput gene-expression analysis using quantitative real-time PCR and specific designed arrays; (2) “In silico” analysis to identify the potential molecular components of the inflammasome-system regulated by the intervention; and, (3) validation studies at gene expression level in PBL and at secreted protein level in plasma. Analysis will be performed by RT-PCR (gene expression) and immunoassays (protein level).

In silico analysis will be performed using different public database and the Ingenuity Pathway Analysis (IPA; http://www.ingenuity.com/) and microRNA target predictions by calculating the minimum free energy (MFE) for miRNA-gene interactions. For Statistical analysis we will use the software Stata and the language R.

Timetable
The study is planned to be accomplished within a 12 month period (finished by December 2021). Transcriptomic studies (point 1) will be started at month 1 and will run over 3-4 months. In silico studies will take approximately 2 months after finishing the transcriptomic analysis and thereafter, we will perform the validation studies (2-4 months). Final statistics and manuscript preparation will be accomplished in 2 months.

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