Inhibitory effects of Levilactobacillus brevis IBRC-M10790 on apoptosis and inflammation induced by Clostridioides difficile culture supernatant in vitro

Abstract

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea that contributes significantly to global morbidity and mortality. Bacterial virulence factors, mostly toxins, play key roles in CDI pathogenesis. Probiotic supplementation is a potential strategy to reduce the adverse effects of C. difficile and support intestinal homeostasis. This study aimed to investigate the inhibitory effects of live Levilactobacillus brevis IBRC-M10790 (LLB) and its membrane vesicles (LBMVs) on apoptosis and inflammation induced by released C. difficile virulence factors in vitro. We employed human colorectal adenocarcinoma Caco-2 and HT-29 cell lines, which are widely used as in vitro models against CDI. Viability and apoptosis of both cell lines were assessed using MTT and Annexin V/PI flow cytometry assays. Anti-inflammatory and anti-apoptotic effects of LLB and LBMVs were investigated following treatment with cell-free supernatants of toxigenic C. difficile RT001 (Tox-S), as well as the culture filtrates of non-toxigenic C. difficile RT084 and ATCC 700057 strains. The expression of apoptosis-related genes (BAX, BCL-2, Caspase-3, Caspase-9) and inflammatory markers (IL-6, IL-8, IL-1β, TNF-α) was measured by RT-qPCR, and cytokine production was analyzed by ELISA. C. difficile Tox-S and culture filtrate significantly reduced cell viability and increased the expression of apoptotic and proinflammatory markers in Caco-2 and HT-29 cells. LLB and LBMVs effectively modulated cell viability, reduced apoptosis, and downregulated the expression and production of inflammatory cytokines in both cell lines after exposure to C. difficile culture supernatants. These findings suggest that LLB and LBMVs could be exploited as potential supplement to the current treatment strategies against C. difficile-induced cellular injury and inflammation.

Keywords: Clostridioides difficile infection; Levilactobacillus brevis; Apoptosis; Inflammation; Membrane vesicles; Tox-S.

Fig. 1

The effects of Tox-S and culture filtrates of C. difficile strains, LLB and LBMVs on apoptosis-related gene expression in Caco-2 and HT-29 cells. Data represents relative gene expression of BCL-2 (A), BAX (B), Caspase-3 (C), and Caspase-9 (D) measured by RT-qPCR in Caco-2 and HT-29 cells after 24 h treatment with Tox-S derived from the toxigenic C. difficile RT001 (100 µg/ml), culture filtrates of the non-toxigenic RT084 (100 µg/ml) and ATCC 700057 (500 µg/ml) strains, LLB (MOI 100), or LBMVs (100 ng/ml). Gene expression data were normalized to β-actin as the reference gene. Data are presented as mean ± SD from three independent experiments. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. Comparisons were made between each treatment group and the untreated control group, and between Tox-S or culture filtrate-treated groups and co-treatment groups with LLB or LBMVs. A P value of < 0.05 was considered significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) by one-way ANOVA statistical analysis. LLB, live Levilactobacillus brevis; LBMVs, Levilactobacillus brevis membrane vesicles.

Fig. 2

The protective effects of LLB and LBMVs on C. difficile Tox-S and culture filtrates induced apoptotic gene dysregulation in Caco-2 and HT-29 cells. Relative expression of BCL-2 (A), BAX (B), Caspase-3 (C), and Caspase-9 (D) in Caco-2 and HT-29 treated with Tox-S of C. difficile RT001 (100 µg/ml), culture filtrates of RT084 (100 µg/ml) and ATCC 700057 (500 µg/ml) strains, LLB, and LBMVs. Gene expression data were normalized to β-actin as the reference gene. Data are presented as mean ± SD from three independent experiments. Statistical comparisons were made as described in Fig. 1. LLB, live Levilactobacillus brevis; LBMVs, Levilactobacillus brevis membrane vesicles.

Fig. 3

The inhibitory effects of LLB and LBMVs on C. difficile Tox-S and culture filtrates induced apoptosis in Caco-2 cells determined by Flow cytometry. Flow cytometry by Annexin V-FITC/PI staining was used to determine the proportion of apoptosis in Caco-2 cells after 24 h treatment with the indicated groups. Density plots show necrotic (Q1), late apoptotic (Q2), early apoptotic (Q3), and viable cells (Q4). Treatment of Caco-2 cells with C. difficile Tox-S or culture filtrates exposed to LLB and LBMVs resulted in reduced apoptosis (A-L). The bar graphs represent percentages of early (M), late (N), and total (O) apoptotic cells. Data are presented as mean ± SD from three independent experiments. Statistical comparisons were made as described in Fig. 1. LLB, live Levilactobacillus brevis; LBMVs, Levilactobacillus brevis membrane vesicles.

Fig. 4

The effects of Tox-S and culture filtrates of C. difficile strains, LLB and LBMVs on proinflammatory cytokine expression and production in Caco-2 and HT-29 cells. Relative gene expression of IL-8 (A), IL-6 (B), IL-1β (C), and TNF-α (D) in Caco-2 and HT-29 cells was measured by RT-qPCR after treatment with Tox-S of C. difficile RT001 (100 µg/ml), culture filtrates of RT084 (100 µg/ml) and ATCC 700057 (500 µg/ml) strains, LLB (MOI 100), and LBMVs (100 ng/ml) overnight. The production of proinflammatory cytokines IL-8 (E), IL-6 (F), IL-1β (G), and TNF-α (H) in culture supernatants was assessed by ELISA. Data are presented as mean ± SD from three independent experiments. Statistical comparisons were made as described in Fig. 1.

Fig. 5

The protective effects of LLB and LBMVs on C. difficile Tox-S and culture filtrates induced proinflammatory response in Caco-2 and HT-29 cells. Relative gene expression of IL-8 (A), IL-6 (B), IL-1β (C), and TNF-α (D) assessed by RT-qPCR, and cytokine secretion levels of IL-8 (E), IL-6 (F), IL-1β (G), and TNF-α (H) determined by ELISA after treatment with Tox-S of C. difficile RT001 (100 µg/ml), and culture filtrates of RT084 (100 µg/ml) and ATCC 700057 (500 µg/ml) strains. Data are presented as mean ± SD from three independent experiments. Statistical comparisons were made as described in Fig. 1. LLB, live Levilactobacillus brevis; LBMVs, Levilactobacillus brevis membrane vesicles.

Fig. 6

Proposed mechanisms by which LLB and LBMVs alleviate apoptosis and inflammation in epithelial cells exposed to C. difficile released virulence factors. Treatment with LLB and LBMVs modulates apoptosis by upregulating anti-apoptotic gene (BCL-2) and downregulating pro-apoptotic genes (BAX, Caspase-3, Caspase-9), and attenuates inflammation by reducing IL-8, IL-6, and TNF-α expression. LLB, live Levilactobacillus brevis; LBMVs, Levilactobacillus brevis membrane vesicles.