AUTHOR=Bueno Manuela R. , Ishikawa Karin H. , Almeida-Santos Gislane , Ando-Suguimoto Ellen S. , Shimabukuro Natali , Kawamoto Dione , Mayer Marcia P. A. 

TITLE=Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.846192

DOI=10.3389/fmicb.2022.846192

ISSN=1664-302X

ABSTRACT=<p>Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This <italic>in vitro</italic> study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by <italic>Aggregatibacter actinomycetemcomitans</italic>. OBA-9 GECs were infected with <italic>A. actinomycetemcomitans</italic> strain JP2 at an MOI of 1:100 and/or co-infected with <italic>Lactobacillus acidophilus</italic> La5 (La5) or <italic>Lacticaseibacillus rhamnosus</italic> Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1β, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with <italic>A. actinomycetemcomitans</italic> or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of <italic>A. actinomycetemcomitans</italic> to GECs was 1.8 × 10<sup>7</sup> (± 1.2 × 10<sup>6</sup>) cells/well in the mono-infection but reduced to 1.2 × 10<sup>7</sup> (± 1.5 × 10<sup>6</sup>) in the co-infection with Lr32 and to 6 × 10<sup>6</sup> (± 1 × 10<sup>6</sup>) in the co-infection with La5 (p &lt; 0.05). GECs mono-infected with <italic>A. actinomycetemcomitans</italic> produced CXCL-8, GM-CSF, and IL-1β, and the co-infection with both probiotic strains altered this profile. While the co-infection of <italic>A. actinomycetemcomitans</italic> with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1β. The probiotics upregulated the expression of <italic>TLR2</italic> and downregulated <italic>TLR4</italic> in cells co-infected with <italic>A. actinomycetemcomitans</italic>. <italic>A. actinomycetemcomitans-</italic>induced the upregulation of <italic>NRLP3</italic> was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene <italic>BCL-2</italic> was upregulated, whereas the pro-apoptotic <italic>BAX</italic> was downregulated in cells co-infected with <italic>A. actinomycetemcomitans</italic> and the probiotics. Infection with <italic>A. actinomycetemcomitans</italic> induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in <italic>A. actinomycetemcomitans</italic> colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, <italic>L. acidophilus</italic> La5 properties suggest that this strain has a higher potential to control <italic>A. actinomycetemcomitans-</italic>associated periodontitis than <italic>L. rhamnosus</italic> Lr32.</p>