Effect of probiotic Lactobacillus rhamnosus by-products on gingival epithelial cells challenged with Porphyromonas gingivalis
Introduction
Probiotics improve health potentially through many mechanisms (Rolfe, 2000), including increasing epithelial barrier function (Ewaschuk et al., 2008) and modulation of the immune response (Isolauri, Sütas, Kankaanpää, Arvilommi, & Salminen, 2001; Lai et al., 2010). There has been increasing interest in probiotics control of periodontal diseases, and clinical trials have been conducted to elucidate their possible impact on oral health (Matsubara, Bandara, Ishikawa, Mayer, & Samaranayake, 2016). Plausible mechanisms of action for probiotics in periodontal diseases are based on modifications of the pathogenic potential of biofilm and include interference on growth and development of periodontal pathogens, the replacement of pathogenic microorganisms by beneficial bacteria, and prevention of colonization by periodontopathogens (Yanine et al., 2013).
The use of probiotics could be an alternative to the overuse of antibiotics, with no concerns regarding drug resistance (Bonifait, Chandad, & Grenier, 2009). Probiotic lactobacilli are very heterogeneous and we have shown that they can elicit distinct response in P. gingivalis infected gingival epithelial cells. Several tested lactobacilli were able to inhibit the adhesion to and invasion of P. gingivalis in gingival epithelial cells (GECs), but unexpectedly, the strain L. rhamnosus Lr-32 triggered GECs leading to increased production of IL-1β by P. gingivalis infected cells (Albuquerque‐Souza et al., 2019).
L. rhamnosus is frequently isolated from dairy products and humans, and most strains, especially the human isolates, display anti-microbial activity against pathogens (Douillard et al., 2013). The probiotic strain L. rhamnosus Lr-32 has been studied for human gastrointestinal affections and is considered safe for human consumption, inhibits pathogens such as Staphylococcus aureus and E. coli in vitro, and is able to modulate immune function (Memorandum, 2008). L. rhamnosus Lr-32 beneficial effect may be extended to the control of candidiasis since its administration as a cheese additive decreased Candida albicans colonization levels in dental wearers (Miyazima, Ishikawa, Mayer, Saad, & Nakamae, 2017). Although the mechanisms underlying the in vivo effect were not completely understood, in vitro data indicated that L. rhamnosus Lr-32 increased IL-1β and IL-10 production, and decreased IL-12 levels in THP1-macrophages with or without Candida albicans/and or lipopolysaccharide (LPS) challenge, with concomitant decreased expression of the receptors dectin-1 and TLR4 (tool-like-receptor 4) (Matsubara et al., 2017). Furthermore, L. rhamnosus Lr-32 reduced the transition of C. albicans from the commensal yeast form to the invasive hypha form in a biofilm in vitro model (Matsubara, Wang, Bandara, Mayer, & Samaranayake, 2016).
Considering L. rhamnosus Lr-32 potential use, and its intriguingly excessive proinflammatory profile, we evaluated the effects of its products on GECs infected with P. gingivalis. Published evidence suggests that products isolated from probiotics, termed postbiotics, would be as effective as probiotics (Adams, 2010; Kataria, Li, Wynn, & Neu, 2009; Zakostelska et al., 2011). Postbiotics comprise enzymes, teichoic acids, peptides derived from peptidoglycan, exopolysaccharides, surface and secreted proteins and bacteriocins generated by a probiotic organism (Konstantinov, Kuipers, & Peppelenbosch, 2013; Tsilingiri et al., 2012). These metabolites and signaling molecules exhibit a broad spectrum of antibacterial and immunomodulatory activities (Cicenia et al., 2014). Despite the proven benefits of probiotics, there are many concerns about their side effects such as the spread of antibiotic resistance genes, virulence factors of a given strain, translocation to tissues or blood, risk of sepsis in premature babies and impairment of normal microbiota colonization (Kataria et al., 2009). Postbiotics may also be advantageous over probiotics due to their chemical structure, safety dose parameters and longer shelf life (Shenderov, 2013). Thus, we hypothesized that products of L. rhamnosus Lr-32 (spent media and lysate) altered the gingival epithelial cells response induced by P. gingivalis.
Section snippets
Bacterial strains and growth conditions
The probiotic strain L. rhamnosus Lr-32™(Danisco, Madison, WI, USA) was cultivated under microaerophilic conditions (5% CO2, 37 °C) in broth and agar MRS (Man Rogosa and Sharp, Difco Laboratories, Detroit, MI, USA). P. gingivalis ATCC33277 was grown under anaerobic conditions (90 % N2, 5 % CO2 and 5 % H2, 37 °C) on blood agar plates [Brain Heart agar, Difco Laboratories) enriched with 5 % defibrinated sheep blood, 0.5 mg mL−1 hemin, and 1 mg mL−1menadione] and in BHHM broth [Brain Heart broth
Results
Both P. gingivalis and living probiotic L. rhamnosus Lr-32 did not alter the OBA-9 cell viability compared to control (p > 0.05). The spent media significantly reduced OBA-9 cell viability when compared to control (OBA-9 in KSFM without antibiotic) either used alone or together with P. gingivalis (in both situations: in co-culture with P. gingivalis for 4 h and after prior infection with P. gingivalis for 2 h) (p < 0.05). Cell viability of the other experimental groups did not differ from
Discussion
We investigated the effect of live probiotic L. rhamnosus Lr-32, probiotic lysate and spent culture medium on P. gingivalis-infected OBA-9 cells. Only spent culture medium significantly affected the viability of gingival epithelial cells either alone or in co-culture with P. gingivalis, possibly caused by some byproduct released by the bacteria in the spent media, such as organic acids produced during the probiotic metabolism. Although this is the first study that addresses the by-products of a
Funding information
This project was funded by the São Paulo Research Foundation (FAPESP), grant # 2015/18273-9. GV was supported by a FAPESP scholarship #2017/10829-3.
Data availability
All data generated or analysed during this study are included in this published article.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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