Blood pressure changes correlate with short-chain fatty acid production potential shifts under a synbiotic intervention

Gut microbes produce various metabolites which mediate interactions with host physiology. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are metabolites produced from otherwise indigestible polysaccharides (fibres) through bacterial fermentation in the intestine.1 SCFA supplementation and a high-fibre diets have been shown to influence blood pressure (BP) and hypertensive end-organ damage in experimental models.1–3 Hypertension is characterized by a pro-inflammatory immune response, which leads to the accumulation of damage to the cardiovascular system.4 In humans, inflammatory serum markers such as C-reactive protein are cardiovascular risk markers. Here, we analysed clinical and microbiome sequence data from a recently published study5 (ISRCTN registry ID: ISRCTN37346212), patients had provided informed written consent prior to inclusion in the study and conforming to the principles outlined in the declaration of Helsinki, to assess whether changes in gut microbial SCFA production potential is also associated with BP reduction in human subjects. Our study aimed to assess whether SCFA production capacity may be a useful marker of microbiome functionality, as this role appears to be particularly relevant in cardiovascular disease (CVD). Metagenomic samples underwent standard read trimming, quality control, adapter, and human reads removal, using the MOCAT tool.6 They were mapped to the IGC 10M integrated gut microbial gene catalogue,7 which was mapped to the KEGG database using MOCAT2.6 We quantified the abundance of all KEGG KO gene families, which were then binned to the gut metabolic modules (GMMs) using the GOMixer tool (online tool available and downloadable software package: http://www.raeslab.org/gomixer/), and selected for SCFA metabolism pathways to produce SCFA metabolic potential estimates. These estimates were averaged within each GMM and summed across all modules contributing to production of each respective SCFA. Further details on the samples and their processing, quality control, read statistics, and accessions (ENA: PRJEB17632) were previously published.5 Changes for cardiovascular risk parameters vs. change in SCFA production potentials were tested using Spearman correlation tests. P-values were adjusted for the multiple tests (N = 3 × 3 hypotheses) using Benjamini–Hochberg corrections. A Q-value of <0.1 was considered significant. The extent of change between pre- and post-intervention levels of CVD markers was assessed using Cliff’s delta effect size estimates. We re-analysed data from a recent randomized, double-blind, placebo-controlled trial investigating the effect of a synbiotic intervention on gut microbiome composition and cardiovascular risk markers in n = 84 human subjects with metabolic syndrome (n = 48) and healthy controls (n = 26).5 For 90 days, participants received a twice daily 200 g synbiotic yoghurt which contained live bacteria 108 CFU/daily dose (Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, and Bifidobacterium bifidum) and prebiotic fibres (2% inulin, 0.2% pectin), or a placebo.5 Live bacteria from the synbiotic together with inulin were capable of producing SCFA in vitro (data not shown). Pre- and post-intervention systolic blood pressure (SBP) and diastolic blood pressure (DBP), inflammatory serum marker high-sensitivity C-reactive protein (hsCRP), and gut microbiome composition were recorded. On average, the synbiotic intervention compared to placebo did not significantly change SCFA production potential, hsCRP or BP (data not shown). However, in patients receiving the synbiotic intervention, an increase in SCFA production capacity correlated with a reduction in SBP, DBP, and hsCRP, reaching significance (Spearman test, BH FDR < 0.1) for DBP (Figure 1). Blood pressure and hsCRP correlate with SCFA production capacity under synbiotic intervention. Re-analysis of a placebo-controlled intervention study in (n = 84) participants with and without metabolic syndrome. Patients received either a synbiotic (n = 43) or placebo (n = 41). Changes in SCFA (acetate, butyrate, and propionate) production bacterial gene module abundances (heatmap, horizontal axis) anti-correlate with changes in risk parameters (vertical axis) under synbiotic but not placebo treatment. Correlations for diastolic blood pressure (DBP, mmHg) are significant (Spearman test •BH-FDR<0.1; *BH-FDR<0.05). Serum C-reactive protein (hsCRP, mg/L) and systolic blood pressure (SBP, mmHg) show corresponding trends. Linear regressions for changes in DBP (mmHg, y-axis) and changes in SCFA production potential (acetate, butyrate, and propionate, from left to right, x-axis) below. In experimental models, SCFAs were shown to be beneficial in hypertension.1–3 To obtain preliminary insights on effects of bacterial SCFA production capacity and BP in humans, we re-analysed data from a clinical trial.5 Participants that experienced a decrease in BP upon synbiotic treatment exhibited increased capacities for intestinal SCFA production. Similarly, a decrease of the serum inflammation marker hsCRP was correlated with increased SCFA production capacity post-intervention. This implies that the extent to which this type of intervention can modulate BP and hsCRP is dependent on increased SCFA production capacity in the gut. Therefore, we think SCFA production capacity may be a useful metric to assess the health of the host-microbiome interface in CVD patients. Our analysis included metabolic syndrome patients and healthy controls, suggesting a conserved mechanism relevant in diseased and healthy individuals. Conclusions from the current re-analysis may be limited due to the high variability of the human gut microbiome as well as dietary variability, impacting both composition and activity of the gut microbiota. Further clinical studies are needed and should consider direct measurements of circulating and intestinal SCFA levels in addition to its bacterial production potential. Conflict of interest: none declared. The Deutsche Forschungsgemeinschaft (German Research Foundation) Projektnummer 394046635 - CRC 1365 and the DZHK (German Center for Cardiovascular Research, 81Z1100101) supported to D.N.M., N.W., and S.K.F. N.W. is participant in the Clinician Scientist Program funded by the Berlin Institute of Health (BIH), supported by a grant from the Corona-Foundation (Corona-Stiftung im deutschen Stiftungsverband), and by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (852796).