Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 ameliorate barrier dysfunction in human induced pluripotent stem cell-derived crypt-villus structural small intestine

Abstract

Background: Lactic acid bacteria (LAB) have been widely used as probiotics which contribute to our health. We previously reported that Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, two yogurt starter strains, ameliorate the intestinal barrier dysfunction caused by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. However, Caco-2 cells differ from living organisms in various ways. We have developed a human induced pluripotent stem cell-derived crypt-villus structural small intestine (hiPSC-SI) was established with a villus-like structure containing constituent cells of the small intestine.

Methods: A hiPSC-SI and LAB co-culture model was established to assess the impact of LAB on barrier function and elucidate the underlying mechanisms.

Results: The medium on the luminal side for co-culturing cells and bacteria was examined and determined to use Hanks' balanced salt solution without glucose in terms of bacterial survival rate. LAB were found to ameliorate permeability and decrease the gene expression of tight junction associated proteins induced by TNF-α and IFN-γ. Regarding cell differentiation, LAB suppressed the downregulation of LGR5, VIL1, LYZ and MUC2 by cytokines. Moreover, they ameliorated reduced mucin 2 protein production and decreased the number of mucin 2-positive cells. Finally, transcriptome analysis suggested that they ameliorated the aberration in cytokine-induced cell differentiation via an anti-inflammatory effect on intestinal stem cells.

Conclusions: The results indicate that LAB ameliorate the cytokine-induced dysfunction of intestinal barrier integrity and homeostasis disrupted by cytokines in a co-culture model of hiPSC-SI and LAB.

Keywords: anti-inflammation; barrier function; differentiation; induced pluripotent stem cell-derived crypt-villus structural small intestine; lactic acid bacteria; mucin 2; tight junction.

Figure 1

Culture and experimental protocol for human induced pluripotent stem cell-derived crypt-villus structural small intestine (hiPSC-SI). hiPSC-derived small intestinal organoids were dispersed as single cells and seeded onto ThinCert 24 well cell culture inserts. The cells were cultured in conditioned medium containing various differentiation factors. From day 3, the cells were cultured at the air-liquid interface. From day 8 onwards, the activity of lactic acid bacteria (LAB) on hiPSC-SI was examined. PS, penicillin and streptomycin; Glu, glucose.

Figure 2

The number of Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 was maintained after culturing in HBSS without glucose. (A) L. bulgaricus 2038 and (B) S. thermophilus 1131 were cultured in conditioned medium with or without penicillin and streptomycin (PS (+) or PS (–)) or HBSS with or without glucose (Glu (+) or Glu (–)) anaerobically at 37 °C for 18 h. The colony-forming unit (CFU) percentage of the initial value is shown (n = 6). The dashed line represents 100%. CM, conditioned medium.

Figure 3

HBSS did not change the morphology, barrier function, or intestinal-associated gene expression. In the HBSS Glu (+) and HBSS Glu (–) groups, HBSS with or without glucose was added to the apical side on day 8, respectively. (A) The 3D structure was observed on day 14 (scale bar, 200 µm). (B) The trans-epithelial electrical resistance (TEER) values were measured immediately before medium change. (C) Cells were harvested on day 14 and intestine-associated gene expression was analyzed using real-time polymerase chain reaction. Data were normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression and expressed relative to the adult small intestine as 1. Data are presented as mean ± standard error (n = 3).

Figure 4

Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 improved the intestinal barrier dysfunction induced by TNF-α and IFN-γ. L. bulgaricus 2038, S. thermophilus 1131, TNF-α, and IFN-γ were added to hiPSC-SI on day 8. (A) The trans-epithelial electrical resistance (TEER) percentage of the initial value and (B) the permeability of fluorescein isothiocyanate-dextran with an average molecular weight of 4,000 (FD-4) after 42 h in the experiments are shown (n = 5). (C-L) The gene expression levels of TJ-associated proteins ((C) CLDN1, (D) CLDN3, (E) CLDN4, (F) CLDN7, (G) CLDN12, (H) OCLN, (I) TJP1, (J) TJP2, (K) TJP3, and (L) F11R) were evaluated via real-time polymerase chain reaction. Data were normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression and are shown as relative expression levels (n = 5). Comparisons were performed using Dunnett’s test. *** P< 0.001, ** P< 0.01, * P< 0.05, ^† P< 0.1.

Figure 5

Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 suppressed the gene expression downregulation of cell markers by TNF-α and IFN-γ. L. bulgaricus 2038, S. thermophilus 1131, TNF-α, and IFN-γ were added to the cells. Cells were collected after 42 h in the experiments. The gene expression levels of cell markers ((A) LGR5, (B) VIL1, (C) MUC2, and (D) LYZ) were evaluated via real-time polymerase-chain reaction. Data were normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression and are shown as relative expression levels (n = 5). Comparisons were performed using Dunnett’s test. *** P< 0.001, ** P< 0.01.

Figure 6

Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 ameliorated the decrease in mucin 2 production and mucin 2-positive cells by TNF-α and IFN-γ. L. bulgaricus 2038, S. thermophilus 1131, TNF-α, and IFN-γ were added to the cells. (A) Cells were collected after 42 h in the experiments. The mucin 2 concentration was measured by enzyme-linked immunosorbent assay (n = 5). Comparisons were performed with the Brunner-Munzel test followed by the Benjamini-Hochberg correction. * P< 0.05. (B) Mucin 2-positive cells were detected by immunofluorescence microscopy (scale bar, 20 μm). (C) Gene set enrichment analysis was performed. Enrichment plots for the gene set of REACTOME_O_LINKED_GLYCOSYLATION are shown. Comparisons between the control and TNF-α + IFN-γ groups (left panel), the TNF-α + IFN-γ and TNF-α + IFN-γ + L. bulgaricus 2038 groups (central panel), and the TNF-α + IFN-γ and TNF-α + IFN-γ + S. thermophilus 1131 groups (right panel) are also shown.

Figure 7

Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 activated and suppressed various gene sets. Gene set enrichment analysis was also performed. Enrichment plots for the gene sets of HALLMARK_INFLAMMATORY_RESPONSE are shown. Comparisons between (A) the control and TNF-α + IFN-γ groups, (B) the TNF-α + IFN-γ and TNF-α + IFN-γ + L. bulgaricus 2038 groups, and (C) the TNF-α + IFN-γ and TNF-α + IFN-γ + S. thermophilus 1131 groups are also shown.