Specific Strains of Lactic Acid Bacteria Differentially Modulate the Profile of Adipokines In Vitro

Abstract

Obesity induces local/systemic inflammation accompanied by increases in macrophage infiltration into adipose tissue and production of inflammatory cytokines, chemokines, and hormones. Previous studies have shown that probiotics could improve the intestinal dysbiosis induced by metabolic diseases such as obesity, diabetes, and metabolic syndrome. Microorganisms could (directly or indirectly) affect adipokine levels due to their capacity to induce translocation of several intestinal microbial antigens into systemic circulation, which could lead to metabolic endotoxemia or produce immunomodulation in different organs. The aim of the present study was to select non-inflammatory lactic acid bacteria (LAB) strains with the capacity to modulate adipokine secretion by the adipose tissue. We wish to elucidate the role of potential probiotic strains in the regulation of the cross talking between immune cells such as macrophages and adipose cells. Mouse macrophage cell line RAW 264.7 was used for evaluating the ability of 14 LAB strains to induce cytokine production. The LAB strains were chosen based on their previously studied beneficial properties in health. Then, in murine adipocyte culture and macrophage-adipocyte coculture, we determined the ability of these strains to induce cytokines and leptin secretion. Tumor necrosis factor alpha, interleukin 6 (IL-6), IL-10, monocyte chemoattractant protein-1, and leptin levels were measured in cell supernatants. We also performed the detection and quantification of leptin receptor (Ob-Rb) expression in macrophage cell lines stimulated by these LAB strains. Differential secretion profile of cytokines in macrophage cells induced by LAB strains was observed. Also, the levels of Ob-Rb expression diverged among different LAB strains. In LAB-stimulated coculture cells (adipocytes and macrophages), we observed differential production of leptin and cytokines. Furthermore, we detected lower production levels in single culture than cocultured cells. The principal component analysis showed an association between the four clusters of strains established according to their inflammatory profiles and leptin adipocyte production and leptin receptor expression in macrophages. We conclude that coculture is the most appropriate system for selecting strains with the ability to modulate adipokine secretion. The use of microorganisms with low and medium inflammatory properties and ability to modulate leptin levels could be a strategy for the treatment of some metabolic diseases associated with dysregulation of immune response.

Keywords: adipocyte; adipokine; lactic acid bacteria; leptin; macrophage; probiotics.

Figure 1

Production of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and IL-10 by RAW 264.7 cells stimulated with different lactic acid bacteria (LAB) strains. Cytokines/chemokine and leptin concentrations were assayed in supernatants from LAB-stimulated RAW 264.7 cells. Macrophages were treated for 24 h with bacterial cell suspensions, and TNF-α (A), IL-6 (B), MCP-1 (C), and IL-10 (D) levels were quantified using cytometric bead array kit and leptin levels, using enzyme-linked immunosorbent assay test. Purified lipopolysaccharide from Escherichia coli was used as a positive control. Non-stimulated cells were evaluated as controls of basal cytokine levels (negative control). The stimulating strains were CRL431, CRL258, CRL1063, CRL66, CRL72, CRL117, CRL1446 CRL1434, CRL350, CRL352, CRL353, CRL355, CRL575, and L. CRL576 strains. Results are expressed as mean ± SD of triplicate measures determined in two independent experiments. Mean values in the same graphic with different letters (a–d) were significantly different (P < 0.05). The relationships among strains, cytokines/chemokine wer obtained using (PCA) and Pearson correlation coefficients. In the biplot obtained by PCA (E), points are coded by stimulating strains and controls. The position of some points was slightly modified to avoid overlapping of the labels.

Figure 2

Leptin receptor (Ob-Rb) expression in RAW 264.7 cells. RAW 264.7 cells after lactic acid bacteria (LAB) stimulation were incubated with a polyclonal rabbit antimouse Ob-Rb Ab and a secondary FITC-conjugated Ab (antirabbit IgG). Ten thousand cells were analyzed per sample at an argon laser flow cytometry scanner. (A) Histograms correspond to the analysis of Ob-Rb expression by non-stimulated (negative control) and LAB-stimulated RAW 264.7 cells. The stimulating strains were CRL431, CRL258, CRL1063, CRL66, CRL72, CRL117, CRL1446 CRL1434, CRL350, CRL352, CRL353, CRL355, CRL575, and CRL576. Lipopolysaccharide (LPS) was used as a positive control. Open black line represents fluorescent labeling of Ob-Rb positive cells of negative control, and gray areas represent other strains stimulus. (B) Percentage of Ob-Rb expression in RAW 264.7 cells. The bars represent the different LAB strains. The results are expressed as media of percentages of cells Ob-Rb (+) ± SD of two experiments. Mean values in the same graphic with different letters (a–d) were significantly different (P < 0.05). (C) Microphotograph representative of the Ob-Rb expression in non-stimulated macrophages (negative control, C-1) and LPS-stimulated macrophages culture (positive control, C-2).

Figure 2

Leptin receptor (Ob-Rb) expression in RAW 264.7 cells. RAW 264.7 cells after lactic acid bacteria (LAB) stimulation were incubated with a polyclonal rabbit antimouse Ob-Rb Ab and a secondary FITC-conjugated Ab (antirabbit IgG). Ten thousand cells were analyzed per sample at an argon laser flow cytometry scanner. (A) Histograms correspond to the analysis of Ob-Rb expression by non-stimulated (negative control) and LAB-stimulated RAW 264.7 cells. The stimulating strains were CRL431, CRL258, CRL1063, CRL66, CRL72, CRL117, CRL1446 CRL1434, CRL350, CRL352, CRL353, CRL355, CRL575, and CRL576. Lipopolysaccharide (LPS) was used as a positive control. Open black line represents fluorescent labeling of Ob-Rb positive cells of negative control, and gray areas represent other strains stimulus. (B) Percentage of Ob-Rb expression in RAW 264.7 cells. The bars represent the different LAB strains. The results are expressed as media of percentages of cells Ob-Rb (+) ± SD of two experiments. Mean values in the same graphic with different letters (a–d) were significantly different (P < 0.05). (C) Microphotograph representative of the Ob-Rb expression in non-stimulated macrophages (negative control, C-1) and LPS-stimulated macrophages culture (positive control, C-2).

Figure 3

Production of TNF-α (A), interleukin 6 (IL-6) (B), monocyte chemoattractant protein-1 (MCP-1) (C), IL-10 (D), and leptin (E) by lactic acid bacteria (LAB)-stimulated mice adipocytes. Cytokines/chemokine and leptin concentrations were assayed in supernatants from LAB-stimulated mice adipocytes. Adipocytes were treated for 24 h with bacterial cell suspensions, and TNF-α, IL-6, MCP-1, and IL-10 levels were quantified using Cytometric Bead Array Kit and leptin levels, using enzyme-linked immunosorbent assay test. LPS was used as a positive control. Non-stimulated cells were evaluated as controls of basal cytokine levels (negative control). The stimulating strains were CRL431, CRL258, CRL1063, CRL66, CRL72, CRL117, CRL1446 CRL1434, CRL350, CRL352, CRL353, CRL355, CRL575, and L. CRL576 strains. Results are expressed as mean ± SD of triplicate measures determined in two independent experiments. Mean values in the same graphic with different letters (a–g) were significantly different (P < 0.05).

Figure 4

Production of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), IL-10, and leptin by macrophage/adipocyte coculture stimulated with lactic acid bacteria (LAB) strains. Cytokines/chemokine and leptin concentrations were assayed in supernatants from LAB-stimulated macrophage/adipocyte coculture. Cells were treated for 24 h with bacterial cell suspensions and TNF-α, IL-6, MCP-1, and IL-10 levels were quantified using Cytometric Bead Array Kit and leptin levels, using enzyme-linked immunosorbent assay test. LPS was used as a positive control. Non-stimulated cells were evaluated as controls of basal cytokine levels (negative control). The stimulating strains were CRL431, CRL258, CRL1063, CRL66, CRL72, CRL117, CRL1446 CRL1434, CRL350, CRL352, CRL353, CRL355, CRL575, and L. CRL576 strains. Results are expressed as mean ± SD of triplicate measures determined in two independent experiments. Mean values in the same graphic with different letters (a–i) were significantly different (P < 0.05). Production levels of (A) TNF-α, (B) IL-6, (C) MCP-1, (D) IL-10, and (E) leptin.

Figure 5

Biplots obtained by principal component analysis (PCA) of cytokines/chemokine and leptin production by macrophage/adipocyte coculture associated with expression of the Ob-Rb receptor in macrophages. Levels of TNF-α, IL-6, MCP-1, IL-10, and leptin produced by macrophage/adipocyte cocultures were associated with the expression of Ob-Rb in macrophages in biplots obtained by PCA. Non-stimulated cells were evaluated as controls basal (negative control) of cytokines/chemokine and leptin levels and Ob-Rb expression. LPS was used as a positive control. In the biplots, points are coded by stimulating strains (CRL431, CRL258, CRL1063, CRL66, CRL72, CRL117, CRL1446 CRL1434, CRL350, CRL352, CRL353, CRL355, CRL575, and L. CRL576) and controls. The position of some points was slightly modified to avoid overlapping of the labels.

Figure 6

Possible inmuno- and adipokine modulation of lactic acid bacteria in an in vitro macrophage/adipocyte system.