Effect of probiotic Lactobacillus rhamnosus by-products on gingival epithelial cells challenged with Porphyromonas gingivalis

Highlights

• L. rhamnosus Lr-32 by-products rather than live probiotic alter the response driven by P. gingivalis in gingival epithelial cells.

• Secreted products by the probiotic in the spent medium decreased cell response to P. gingivalis.

• Cell lysate of probiotic increased transcription of inflammatory genes in P. gingivalis infected gingival epithelial cells.

Abstract

Objective

Probiotics are usually given as living cells, but their effects may be also achieved by postbiotics. We hypothesized that probiotics products (spent media and lysate) altered the response induced by P. gingivalis in gingival epithelial cells (GECS).

Methods

Immortalized human OBA-9 GECs (∼2,5 × 10⁵cells/well) were challenged with P. gingivalis ATCC33277, and co-infected with L. rhamnosus Lr-32 for 4 h. L. rhamnosus Lr-32 spent medium or cells lysate was added to GECs co-infected with P. gingivalis. Another set of OBA-9 GECs were first exposed to P. gingivalis ATCC 33277 and then to the living probiotic or probiotic products. Transcription of genes encoding inflammatory mediators (IL-1β, TNF-α, IL-6, and CXCL-8) and receptors (TLR2 and TLR4) were evaluated by RT-qPCR. P. gingivalis growth under L. rhamnosus Lr-32 postbiotics was also evaluated.

Results

L. rhamnosus Lr-32 spent media decreased cell viability, while living cells and cell lysates did not. L. rhamnosus Lr-32 lysate, but not spent media, upregulated transcription of inflammatory mediators (IL-1β, TNF-α, IL-6, and CXCL-8) in GECs infected with P. gingivalis. Transcription of TRL2 was upregulated in all experimental groups compared to control, whereas TLR4 was upregulated by the probiotic or its postbiotics in P. gingivalis infected cells. Spent media and lysates reduced the growth of P. gingivalis.

Conclusion

L. rhamnosus Lr-32 cell components rather than live probiotic enhanced the expression of inflammatory mediators in P. gingivalis infected gingival epithelial cells. The increased potential of Lr-32 cell lysates to promote immune response to the periodontopathogen may favor pathogen elimination but may also lead to additional deleterious effects of the exacerbated inflammation.

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.