Anti-Atherogenic Actions of the Lab4b Consortium of Probiotics In Vitro

Abstract

Probiotic bacteria have many protective effects against inflammatory disorders, though the mechanisms underlying their actions are poorly understood. The Lab4b consortium of probiotics contains four strains of lactic acid bacteria and bifidobacteria that are reflective of the gut of newborn babies and infants. The effect of Lab4b on atherosclerosis, an inflammatory disorder of the vasculature, has not yet been determined and was investigated on key processes associated with this disease in human monocytes/macrophages and vascular smooth muscle cells in vitro. The Lab4b conditioned medium (CM) attenuated chemokine-driven monocytic migration, monocyte/macrophage proliferation, uptake of modified LDL and macropinocytosis in macrophages together with the proliferation and platelet-derived growth factor-induced migration of vascular smooth muscle cells. The Lab4b CM also induced phagocytosis in macrophages and cholesterol efflux from macrophage-derived foam cells. The effect of Lab4b CM on macrophage foam cell formation was associated with a decrease in the expression of several key genes implicated in the uptake of modified LDL and induced expression of those involved in cholesterol efflux. These studies reveal, for the first time, several anti-atherogenic actions of Lab4b and strongly implicate further studies in mouse models of the disease in vivo and in clinical trials.

Keywords: atherosclerosis; foam cells; gene expression; inflammation; macrophages; probiotics; smooth muscle cells.

Figure 1

Migration of human monocytes in response to three different concentrations of Lab4b CM. MCP-1-induced migration of THP-1 monocytes was followed in the presence of either the vehicle or three different concentrations of Lab4b CM as shown (0.5 μg/mL, 1 μg/mL and 1.5 μg/mL). Cells treated with vehicle but in the absence of MCP-1 were also included for comparative purposes. Migration was determined as a percentage of total cells and shown as fold change in migration relative to the vehicle control with MCP-1 (20 ng/mL), which was arbitrarily assigned as 1. Data are mean ± SEM from five independent experiments and statistical analysis was carried out on log transformed data using one-way ANOVA with the Bonferroni post-hoc test (**, p ≤ 0.01; ***, p ≤ 0.001).

Figure 2

Lab4b CM increases TBHP-induced ROS production in human monocytes and macrophages. THP-1 monocytes (A) and macrophages (B) were treated with 50 μM TBHP and either vehicle or three different concentrations of Lab4b CM, as shown (0.5 μg/mL, 1 μg/mL and 1.5 μg/mL). Cells treated with vehicle but in the absence of TBHP were also included for comparative purposes. ROS production is shown as fold change to the vehicle control with TBHP, which was arbitrarily assigned as 1. Data are mean ± SEM from five independent experiments, and statistical analysis was carried out on sine–transformed (A) or log transformed (B) data using one-way ANOVA with the Bonferroni post-hoc test (**, p ≤ 0.01; ***, p ≤ 0.001; NS, not significant).

Figure 3

Lab4b CM modulates several processes associated with foam cell formation in human macrophages. THP-1 macrophages (A,B,D), HMDM (C) or THP-1 macrophage-derived foam cells (E) were used to determine macropinocytosis (A), Dil-oxLDL uptake (B,C), phagocytosis (D), and cholesterol efflux (E). Macropinocysosis was carried out using three different concentrations of Lab4b CM (0.5 μg/mL, 1 μg/mL, and 1.5 μg/mL), whereas, 1.5 μg/mL was used for all the other assays. The values in cells treated with vehicle alone (A–D) or vehicle and apolipoprotein (apo) A1 (10 μg/mL; (E) were arbitrarily assigned as 100%. Data are mean ± SEM from three independent experiments, and statistical analysis was carried out using one-way ANOVA with Dunnett (A; log transformed data) or Bonferroni (E) post-hoc test, an unpaired Student’s t-test (B,C) or unpaired Welch’s t-test (D) (**, p ≤ 0.01; ***, p ≤ 0.001).

Figure 4

Lab4b CM regulates the expression of several key genes implicated in the control of macrophage foam cell formation. THP-1 macrophages (A,C,D), HMDM (B) or THP-1 macrophage-derived foam cells using acetylated LDL (AcLDL, 25 μg/mL; E–I) treatment for 24 h were incubated for 24 h with vehicle or Lab4b CM (1.5 μg/mL). For THP-1 macrophage-derived foam cells (F–I), cells treated with vehicle alone in the absence any AcLDL treatment were also included for comparative purposes. The expression of CD36 (A,B), SRA (C), LPL (D), ABCA1 (E), ABCG1 (F), LXR-α (G), LXR-β (H), and ApoE (I) was determined on purified RNA by real-time quantitative PCR (RT-qPCR), as described in Materials and Methods, and normalised to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. The data are presented as box-and-whisker plots of log2 fold-change in gene expression relative to the vehicle control, where whiskers represent minimum-to-maximum fold-change from three independent experiments. Statistical analysis was performed using an unpaired Welch’s t-test (A,C,D), an unpaired Student’s t-test (B) or one-way ANOVA with the Bonferroni post-hoc test (E–I) (*, p ≤ 0.05; ***, p ≤ 0.001; NS, not significant).

Figure 5

Lab4b CM attenuates proliferation of human monocytes and macrophages. (A–C), THP-1 monocytes were treated with either vehicle or 1.5 μg/mL Lab4b CM and cell numbers counted at days 2, 5, and 7 and represented to the vehicle control, which was arbitrarily assigned as 100%. (D), THP-1 macrophages were treated for 48 h with vehicle or 1.5 μg/mL Lab4b CM and change in proliferation, as assessed by the crystal violet assay, was determined as a percentage relative to the vehicle control, which was arbitrarily assigned as 100%. (E), The supernatant from the same cells was used to assess viability via LDH activity and determined as a percentage of the vehicle control, which was arbitrarily assigned as 100%. (F), The proliferation rate of THP-1 macrophages was determined by following BrdU incorporation following treatment of THP-1 macrophages with vehicle or 1.5 μg/mL Lab4b CM. Proliferation rate was determined as a percentage of vehicle, which was assigned as 100%. Data are mean ± SEM from three independent experiments, and statistical analysis was carried out using an unpaired Student’s t-test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; NS, not significant).

Figure 6

Lab4b CM affects proliferation and migration of HASMC. (A,B), HASMC were treated for 7 days with vehicle or 1.5 μg/mL Lab4b CM and cell proliferation was determined using crystal violet. The supernatant from the same cells was used to determine cell viability by following LDH activity. (C), HASMC were treated with vehicle or 1.5 μg/mL of Lab4b CM for 2 days and the rate of proliferation was determined by BrdU incorporation. The values from Lab4b CM-treated cells are represented in relation to the vehicle control, which was arbitrarily assigned as 100%. (D), Invasion of HASMC in response to PDGF (20 ng/mL) was determined following treatment with vehicle or 1.5 μg/mL of Lab4b CM for 4h. Cells incubated with vehicle in the absence of PDGF were also included for comparative purposes. The number of migrated cells were counted and averaged per five high-powered field and represented as fold-change to the vehicle control with PDGF, which was arbitrarily assigned as 1. In all cases, data are mean ± SEM from three independent experiments with statistical analysis carried out using an unpaired Student’s t-test (A–C) or one-way ANOVA with the Bonferroni post-hoc test (D) (**, p ≤ 0.01; ***, p ≤ 0.001; NS, not significant).