Lactobacillus plantarum-derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro

Abstract

Probiotics offer various health benefits. Lactobacillus plantarum has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteria-derived EVs on human skin is unclear. Herein, we investigated how L. plantarum-derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre- or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, HLA-DRα expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization in vitro, and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions.

Keywords: IL-10; Lactobacillus plantarum; Probiotics; extracellular vesicles; macrophage polarization.

Figure 1.

Purification of EVs from L. plantarum APsulloc. (a) TRPS analysis of ultracentrifugation (UC)- or OptiPrep density UC (Density)-purified LEVs. The purification of LEVs using two UC methods was performed a minimum of three times and representative TRPS results are shown for each method. (b) Protein concentration and particle numbers of purified LEVs. Conc., concentration; No., number. (c) Bio-TEM and cryo-EM image analyses of density-purified LEVs. The outlined LEV images are enlarged and the lipid bilayer indicated by black and white arrows. Black and white arrowheads indicate the potential double membrane of the vesicles. Scale bars, 200 nm.

Figure 2.

LEV treatment enhances the secretion of anti-inflammatory cytokine IL-10 in human skin organ cultures. Human cultured skin tissues were treated twice with vehicle (HBS), LEVs (50 μg/mL), or SEV (50 μg/mL) during a 6-day culture period. Tissues and supernatants were harvested on day six for H&E staining and cytokine array analysis (n = 2; two independent tissue samples). (a) Scheme of bacterial EV treatment of human skin cultures and H&E staining. The outlined area is enlarged. Scale bars, 100 μm. (b) Abnormality of the epidermis was quantified by measuring the ratio of the number of vacuoles (empty space due to cytoplasmic loss) to the total number of cells in the epidermis. Data are expressed as the mean ratio ± SEM of three different images and statistical significance was analysed by one-way ANOVA. ****p < 0.0001; ns, non-significant. (c) Representative immunoblot images of cytokine array analysis. Markedly increased cytokines by LEV and SEV treatments are indicated by red-coloured boxes and arrows. Inflammatory cytokines that are preferentially, and significantly, increased by SEV treatment are indicated by blue-coloured boxes and arrows. Cytokines that are more than 2-fold increased or decreased in LEV- or SEV-treated compared to vehicle-treated groups are shown left or right, respectively, on the basis of NC/PC in the graph (red line for 2-fold increase; green line for 2-fold decrease). The average fold change in LEV- or SEV- treated groups relative to vehicle control was determined by densitometry using ImageJ (https://imagej.nih.gov/ij/). NC/PC, negative control/positive control of the spots in cytokine array.

Figure 3.

LEV treatment induces the differentiation of human monocytic THP1 cells towards the macrophage lineage. (a) Bright-field microscopic images of THP1 cells in culture. THP1 cells in suspension were treated with vehicle (HBS), PMA (10 nM), medium conc. (medium concentrates, 10 μg/mL), or LEVs (10 μg/mL) for 48 h. Magnification, 100 ×. (b) THP1 cells were treated with medium conc. (10 μg/mL), LEVs (10 μg/mL), PMA (10 nM), or density-purified LEVs (density-LEVs, 10 μg/mL) for 48 h. The mRNA expression of activated macrophage-specific genes was analysed by RT-qPCR. Glucose-6-phosphate dehydrogenase (G6PD) was used for normalization. Data are expressed as the mean fold change ± SEM of triplicate measurements and statistical significance was analysed by one-way ANOVA.

Figure 4.

LEV treatment enhances the expression of M2-polarized cell markers. THP1 cells were treated with LEVs (10 μg/mL) for 48 h. (a) mRNA expression of markers specific for M1 or M2 macrophages was analysed by RT-qPCR. G6PD was used for normalization. Statistically significant fold changes (FC) are indicated in different colours according to the increased degree (light grey, 1.5 < FC < 5; medium grey, 5 < FC < 10; dark grey, 10 < FC). -, non-significant. (b) mRNA expression of representative M1 macrophage-specific surface markers. ns, non-significant. (c) mRNA expression of M2 macrophage-specific markers. Data in (a-c) are expressed as the mean fold change ± SEM of triplicate treatments and statistical significance was analysed by Student’s t-tests.

Figure 5.

Gene expression profiling of secretory proteins after LEV treatment reveals a preferential differentiation of THP1 cells to M2b macrophages. THP1 cells were pre-incubated with PMA (10 nM) for 48 h and treated with vehicle (HBS) or LEVs (10 μg/mL) for an additional 48 h. (a) mRNA expression of M1- and M2-specific chemokines was analysed by RT-qPCR. G6PD was used for normalization. Statistically significant fold changes (FC) are indicated in different colours according to the increased degree (light grey, 1.5 < FC < 5; medium grey, 5 < FC < 10). -, non-significant. (b, c) mRNA expression of representative M1 (b) and M2b (c) macrophage-specific chemokines. ns, non-significant. (d) mRNA expression of M2 macrophage-specific cytokines as determined by RT-qPCR. Data in (a-d) are expressed as the mean fold change ± SEM of triplicate treatments and statistical significance was analysed by Student’s t-tests.

Figure 6.

LEV treatment downregulates inflammation-induced expression of the M1 macrophage cell-surface marker, HLA-DRα. Human THP1 monocytes at day 1 post plating were pre-incubated with 10 nM PMA. PMA was maintained during the treatment period (A) or washed out after 48 h (b). On days 3 and 5, the cells were treated with LEVs (10 μg/ml) or 20 ng/ml of IFN-γ and 10 pg/ml of LPS for 48 h. On day 7, the cells were harvested and HLA-DRα mRNA expression was analysed by RT-qPCR. G6PD was used for normalization. Data are expressed as the mean fold change ± SEM of triplicate measurements and statistical significance was analysed by one-way ANOVA.

Figure 7.

Bacterial fractions prepared under high pressure contain LEV-like nanovesicles and show similar effects as LEVs in human skin organ cultures. (a) Bio-TEM image of LFLs (left) and TRPS analysis for mean diameter and particle number of LFLs (right). The outlined LFL images (1–4) are enlarged and additional large vesicles (5, 6) are shown. Scale bars, 200 nm. (b) Images of H&E staining of human skin cultures after LFL treatment. The cultured tissues were treated with vehicle, LEVs, or LFLs (each 50 μg/ml) twice over a 6-day culture period as indicated in Figure 2a. (c) Abnormality of the epidermis was quantified by measuring the ratio of the number of vacuoles (empty space due to cytoplasmic loss) to the total number of cells in the epidermis. Data are expressed as the mean ratio ± SEM of three different images and statistical significance was analysed by one-way ANOVA. Ns, non-significant. (d, e) Cytokine array analysis of culture supernatants and representative blot image. Markedly increased cytokines by LFL treatment are indicated by red-coloured arrows and boxes. Cytokines that were increased by more than 2-fold in LFL-treated compared to vehicle-treated groups or decreased by 0.5-fold or less in LEV-treated but increased in LFL-treated compared to vehicle-treated groups were shown left or right, respectively, on the basis of NC/PC in the graph (red line for 2-fold increase; green line for 2-fold decrease). The average fold change in LFL-treated groups relative to vehicle control was determined by densitometry using ImageJ. NC/PC, negative control/positive control of the spots in cytokine array.