[Abstract] Dark chocolate (70% cacao) effects human gene expression: Cacao regulates cellular immune response, neural signaling, and sensory perception

Abstract

Dark chocolate (70% Cacao) is a major source of flavonoid antioxidants, which appear to support cognitive, endocrine, and cardiovascular health benefits. However, the in vivo effects of cacao on human genome function are unknown. In a pilot feasibility experimental trial, we examined the effects acute and chronic cacao consumption on human gene expression in circulating blood cells.

Purpose

Map the impact of 70% cacao consumption on human immune and dendritic cell gene expression, with focus on pro/anti-inflammatory cytokines.

Methods

This 8-day (d) pilot study enrolled 4 healthy experimental subjects (2 female and 2 male, age 25–50) and one control (female, 28), who were not taking prescribed or OTC medications. Subjects abstained from all other high-antioxidant foods or supplements 48 h prior to and during the study period. After a fasting baseline blood sample, experimental subjects consumed 48 g 70% cacao (Parliament Chocolates, Redlands, CA) over 10 min (antioxidant activity 46,000 μmoles TE/100g). Follow-up blood samples were obtained 2 h later (acute effects) and 1 wk later (chronic effects). During waking hours (14 h/d) experimental subjects consumed 48 g 70% cacao per day (2 pieces/hr; antioxidant activity 3834 μmoles TE/100g). Total RNA was assayed by RNAseq, with data analyzed by linear models to identify differentially expressed genes, followed by bioinformatic analyses of transcription control pathways and cellular origins.

Results

Leukocyte transcriptome profiles showed little systematic change in the initial 2 h of Cacao exposure, although pre-specified bioinformatics analyses indicated small increases in activity of genes regulated by NF-kB and AP1 transcription factors and CD4+ and CD8+ T cells (all, p < .05). By contrast, analyses of change from baseline to 1 wk identified 177 genes showing significant differential expression using  a priori -specified False Discovery Rate cut-point of .20 (140 up-regulated, 37 down-regulated), with 116 achieving q < .05. Up-regulated genes were involved in leukocyte activation and motility (e.g., CD40, TICAM2, AZU1, HRAS and RASD2, HSPA1L, FGF22); derived from CD4+ and CD8+ T lymphocytes (p < .001 and .0148, respectively); and showed over-representation of promoter elements for NF-kB, SP-1, MZF-1, and HIF-1 transcription factors (all p < .01) and under-representation of AP-1 (p = .018). Up-regulated transcripts also included genes involved in neural signaling and sensory perception (e.g., OR2J3, OR52A5, OR6C3, OR9I1, NR4A2, and NRXN2).

Conclusion

Cacao consumption up-regulates multiple intracellular signaling pathways involved in T cell activation and cellular immune response. These effects emerged weakly within 2 h but there was greater gene expression by the end of 1 wk intervention. Results are consistent with previous indications that cacao components inhibit MAPK activation, but suggest T cell function may be preserved and enhanced via other compensatory molecular pathways. It should be noted that the NF-kB up-regulation source was not from up-regulated monocytes suggesting a role other than cascading inflammation. It is of interest, to note, that genes involved in neural signaling and sensory processes were up-regulated. The functional significance of these effects for immune cells remains to be defined by future expansive research, as do the neurobiological mechanisms involved.