Abstract

Helicobacter pylori infection is a major cause of chronic gastritis and peptic ulcer and a risk factor for gastric malignancies. Although antibiotics-based eradication is effective, this treatment is expensive and causes side effects and antibiotic resistance. Probiotics could present a low-cost, large-scale alternative solution to prevent or decrease the colonization of this gastric pathogen. In fact, some clinical trials have demonstrated that probiotics are effective as adjunct treatment in H. pylori infections and prophylaxis, although deeper studies are necessary to fully understand the mechanisms of action involved in their beneficial effects. In this regard, we have performed a detailed characterization of the probiotic properties and the anti-H. pylori activity of Lactobacillus fermentum UCO-979C isolated from human stomach. We demonstrated that the UCO-979C strain is able to tolerate acid and bile salts; produces hydrogen peroxide; and has high hydrophobicity properties. In addition, we observed that it is able to efficiently adhere to gastric mucosa as demonstrated by in vitro (gastric adenocarcinoma human (AGS) cells) and in vivo (Mongolian Gerbils) experiments. Moreover, we found that L. fermentum UCO-979C strongly inhibited the adhesion, growth and urease activity of H. pylori (Garcia-Cancino et al, 2017 Electronic J Biotechnol 25:75-83). Preliminary studies in AGS cells also showed that L. fermentum UCO-979C significantly reduced H. pylori-induced IL-8 production. However, no deeper immunological studies were performed with this strain. Therefore, the aim of this work was to analyze whether L. fermentum UCO-979C beneficially modulates the innate immune response triggered by H. pylori infection in human gastric epithelial cells and macrophages. The capacity of L. fermentum UCO-979C to modulate immune responses in AGS cells and human THP-1 macrophages was evaluated. For this purpose, AGS cells or PMA-differentiated THP-1 macrophages were treated with different doses of L. fermentum UCO-979C (105, 106 or 107 cells/ml) and then challenged with H. pylori ATCC43504 (107 cells/ml). Cellular damage and levels of TNF-α, IL-1β, IL-6, TGF-β, CCL20 and IL-8 were determined 24 hours after the challenge in AGS cells. Cellular damage and levels of TNF-α, IL-1β, IL-6, IL-10, IL-12p70, IL- 12p40 and IFN-γ were determined 24 hours after the challenge in THP-1 macrophages. It was observed that L. fermentum UCO-979C is able to protect AGS cells against H. pyloriinduced inflammatory damage in a dose dependent manner. A higher viability (MTT-%: UCO979C=95, control=73) and a decreased cytotoxicity (LDH-UI/L: UCO979C=56.4±3.7, control=125.7±4.6) was observed in lactobacilli-treated AGS cells infected with H. pylori when compared to the infected control. In addition, a significant decrease in the production of proinflammatory cytokines and chemokines (IL-1β, IL-6, CCL20 and IL-8) and an increase in the immunoregulatory cytokine TGF-β was observed in UCO-979C-treated cells when compared to the infected control. The most notorious effects were achieved by the smaller dose of L. fermentum UCO-979C (TNF-α-pg/mL: UCO979C=73.2±3.6, control=195.8±3.6; TGF-β-pg/mL: UCO979C=195.7±5.9, control=255.9±5.5). L. fermentum UCO-979C was also able to differentially modulate the inflammatory response triggered by H. pylori in THP1 macrophages. A decreased cytotoxicity (LDH-UI/L: UCO979C=167.3±2.5, control=235.9±3.6) and a higher viability (MTT-%: UCO979C=93, control=84) were detected in UCO-979C-tretaed THP1 macrophages after the challenge with the pathogen when compared to the infected control. The inflammatory cytokines TNF-α and IL-1β were significantly lower in lactobacilli-treated macrophages than in controls after H. pylori challenge (TNF-α-pg/mL: UCO979C=316.5±3.2, control=456.7±3.3). On the contrary, the Th1 cytokines IL-12p70, IL-12p40 and IFN-γ and the immunoregulatory cytokine IL-10 were significantly higher in UCO-979C-treated THP1 cells than in control cells after H. pylori challenge (IFN-γ-pg/mL: UCO979C=342.9±3.4, control=291.4±2.3; IL-10-pg/mL: UCO979C=456.8±3.1, control=325.7±3.5). These findings provide evidence of the beneficial effects of L. fermentum UCO-979C against the inflammatory damage induced by H. pylori infection in both epithelial and immune cells. Although our findings should be proven in appropriate experiments in vivo, in both H. pylori infection animal models and human trials, the results of the present work provide a scientific rationale for the use of L. fermentum UCO-979C to prevent or reduce H. pylori-induced gastric inflammation in humans.