Phytate Hydrolysate Differently Modulates the Immune Response of Human Healthy and Cancer Colonocytes to Intestinal Bacteria

Abstract

(1) Phytic acid (PA) is a component of cereal seeds and legumes, therefore its consumption is much higher in a vegan and vegetarian diet compared to a conventional diet. The diet is the main driver of metabolic activity of gut microbiota, therefore, the ability to degrade phytates by the microbiota of vegans significantly exceeds that of the gut microbiota of omnivores. The aim of the study was to investigate the early phase of the immune response of colonocytes treated with an enzymatic hydrolysate of phytic acid (hPA120) and gut bacteria. (2) Cell lines derived from healthy (NCM460D) and cancer (HCT116) colonic tissue and fecal bacteria from vegan (V) and omnivorous (O) donors were investigated. Fecal bacteria were grown in mucin and phytic acid supplemented medium. Cultured bacteria (BM) were loaded onto colonocytes alone (V BM and O BM) or in combination with the phytate hydrolysate (V BM + hPA120 and O BM + hPA120). After a treatment of 2 h, bacterial adhesion, secretion of cytokines, and the expression of genes and proteins important for immune response were determined. (3) All bacteria-treated colonocytes increased the expression of IL8 compared to controls. The significant increase of the secreted IL-8 (p < 0.01) in both cell lines was observed for O BM and O BM + hPA120. The increase of TNF, IL-1β, and IL-10 secretion in healthy colonocytes (V BM alone and with hPA120 treatments; p < 0.05) and for TNF and IL-10 in cancer cells (treatments except O BM + hPA120 and V BM, respectively; p > 0.05) were stated. A comparison of solely the effect of hPA120 on bacteria-treated colonocytes (BM vs. BM + hPA120) showed that hPA120 decreased expression of NFkB1 and TNFR (p < 0.001) in healthy colonocytes. In cancer colonocytes, the expression of TLR4 and IL1R increased after BM + hPA120 treatment, whereas the secretion of IL-8 and MYD88 and TNFR expression decreased (p < 0.01). (4) The investigated hPA120 showed a differentiated modulatory activity on the immune response of healthy and cancer human colonocytes. Especially when analyzed independently on the gut bacteria origin, it reduced the proinflammatory response of HCT116 cells to gut bacteria, while being neutral for the bacteria-treated healthy colonocytes.

Keywords: colonocytes; conventional diet; gut microbiota; immune response; inositol phosphates; phytate hydrolysate; vegan diet.

Figure 1

Experimental design.

Figure 2

Percentage of adherent bacterial cells (%ADH) to the healthy (NCM460D) and cancer (HCT116) colonocytes. Fecal bacteria derived from vegan (V) and omnivorous (O) volunteers were grown in Wilkins–Chalgren medium supplemented with 0.5% of mucin and 1 mM of phytic acid for 42 h at 37 °C in an anaerobic atmosphere. The adhesion assay was performed with bacteria alone (BM) or with bacteria and the hydrolysate of phytic acid (BM + hPA120), for 2 h at 37 °C. Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median; black dot—outliers. Significant differences were calculated with the Mann–Whitney U test.

Figure 3

Gene expression in NCM460D cells treated with fecal bacteria derived from vegan (V) or omnivorous (O) volunteers. Fecal bacteria were grown in Wilkins–Chalgren medium supplemented with 0.5% of mucin and 1 mM of phytic acid for 42 h at 37 °C in an anaerobic atmosphere. The treatment was performed with bacteria alone (BM) or with bacteria and the hydrolysate of phytic acid (BM + hPA120), for 2 h at 37 °C. The controls were: non-treated colonocytes (CTRL), colonocytes treated with hPA120 and colonocytes treated with non-inoculated microbiological medium treated as bacterial cultures (CTRL-P). Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median; circles—outliers. Kruskal–Wallis was applied to calculate significant differences between the treatments, which are marked with: a—values different at p < 0.05; b—values different at p < 0.01; c—values different at p < 0.001.

Figure 4

Protein expression of TLR4, MyD88 and NFκB p65 in (a)—healthy (NCM460D) and (b)—cancer (HCT116) colonocytes. Fecal bacteria derived from vegan (V) and omnivorous (O) volunteers were grown in Wilkins–Chalgren medium supplemented with 0.5% of mucin and 1 mM of phytic acid for 42 h at 37 °C in an anaerobic atmosphere. The treatment was performed with bacteria alone (BM) or with bacteria and the hydrolysate of phytic acid (BM + hPA120), for 2 h at 37 °C. The controls were: non-treated colonocytes (CTRL), colonocytes treated with hPA120 and colonocytes treated with non-inoculated microbiological medium treated as bacterial cultures (CTRL-P). Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median.

Figure 5

Secretion of interleukin 8 (IL-8), tumor necrosis factor (TNF), interleukin 1β (IL-1 β) and interleukin 10 (IL-10) by healthy (NCM460D) and cancer (HCT116) colonocytes. CTRL—non-treated (control) colonocytes; hPA120—colonocytes treated with phytate hydrolysate; CTRL-P—colonocytes treated with non-inoculated microbiological medium processed as the inoculated one; BM—colonocytes treated with fecal bacteria cultured in Wilkins–Chalgren medium supplemented with 0.5% of mucin and 1 mM of phytic acid for 42 h at 37 °C in an anaerobic atmosphere; BM + hPA120—colonocytes treated with the mixture of BM and hPA120. V or O—fecal bacteria derived from vegan or omnivorous volunteers, respectively. Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median; circles—outliers. Statistical significance was calculated with the non-parametric Kruskal–Wallis test; a—values different at p ≤ 0.05; b—values different at p ≤ 0.01, c—values different at p ≤ 0.001.

Figure 6

Gene expression in HCT116 cells treated with fecal bacteria derived from vegan (V) or omnivorous (O) volunteers. Fecal bacteria were grown in Wilkins–Chalgren medium supplemented with 0.5% of mucin and 1 mM of phytic acid for 42 h at 37 °C in an anaerobic atmosphere. The treatment was performed with bacteria alone (BM) or with bacteria and the hydrolysate of phytic acid (BM + hPA120), for 2 h at 37 °C. The controls were: non-treated colonocytes (CTRL), colonocytes treated with hPA120 and colonocytes treated with non-inoculated microbiological medium treated as bacterial cultures (CTRL-P). Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median; circles—outliers. Kruskal–Wallis was applied to calculate significant differences between the treatments, which are marked with: a—values different at p < 0.05; b—values different at p < 0.01; c—values different at p < 0.001.

Figure 7

Secretion of interleukin 8 (IL-8) by cancer (HCT116) and healthy (NCM460D) human colonocytes after 2-h incubation with fecal bacterial cultures grown in Wilkins–Chalgren medium supplemented with mucin (BM) and phytate. Colonocytes were incubated with BM alone or BM and phytic acid hydrolysate (BM + hPA120). Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median; circles—outliers. Values different at p < 0.01 (Mann–Whitney U test) are marked with “b”.

Figure 8

Comparison of the effect of the phytate hydrolysate (hPA120) on gene expression in bacteria-treated (a)—healthy (NCM460D) and (b)—cancer (HCT116) colonocytes. Fecal bacteria derived from vegan or omnivorous volunteers were grown in Wilkins–Chalgren medium supplemented with 0.5% of mucin and 1 mM of phytic acid for 42 h at 37 °C in an anaerobic atmosphere. Colonocytes were treated with bacteria alone (BM) or with bacteria and the hydrolysate of phytic acid (BM + hPA120), for 2 h at 37 °C. Whiskers—non-outliers, box—25–75% non-outliers; horizontal line in the box—median; circles—outliers. Mann–Whitney U test was applied to calculate significant differences, which are marked with: a—values different at p < 0.05, b—values different at p < 0.01 or c—values different at p < 0.001.