Lactobacillus rhamnosus could inhibit Porphyromonas gingivalis derived CXCL8 attenuation

Ayşegül Mendi, Sevil Köse, Duygu Uçkan, Gülçin Akca, Derviş Yilmaz, Levent Aral, Sibel Elif Gültekin, Tamer Eroğlu, Emine Kiliç, Sina Uçkan, Ayşegül Mendi, Sevil Köse, Duygu Uçkan, Gülçin Akca, Derviş Yilmaz, Levent Aral, Sibel Elif Gültekin, Tamer Eroğlu, Emine Kiliç, Sina Uçkan

Abstract

An increasing body of evidence suggests that the use of probiotic bacteria is a promising intervention approach for the treatment of inflammatory diseases with a polymicrobial etiology. P. gingivalis has been noted to have a different way of interacting with the innate immune response of the host compared to other pathogenic bacteria, which is a recognized feature that inhibits CXCL8 expression.

Objective: The aim of the study was to determine if P. gingivalis infection modulates the inflammatory response of gingival stromal stem cells (G-MSSCs), including the release of CXCL8, and the expression of TLRs and if immunomodulatory L. rhamnosus ATCC9595 could prevent CXCL8 inhibition in experimental inflammation.

Material and methods: G-MSSCs were pretreated with L. rhamnosus ATCC9595 and then stimulated with P. gingivalis ATCC33277. CXCL8 and IL-10 levels were investigated with ELISA and the TLR-4 and 2 were determined through flow cytometer analysis.

Results: CXCL8 was suppressed by P. gingivalis and L. rhamnosus ATCC9595, whereas incubation with both strains did not abolish CXCL8. L. rhamnosus ATCC9595 scaled down the expression of TLR4 and induced TLR2 expression when exposed to P. gingivalis stimulation (p<0.01).

Conclusions: These findings provide evidence that L. rhamnosus ATCC9595 can modulate the inflammatory signals and could introduce P. gingivalis to immune systems by inducing CXCL8 secretion.

Conflict of interest statement

CONFLICT OF INTERESTS

Authors declare no conflict of interests.

Figures

Figure 1. Gingival stromal stem cells (G-MSSCs)…
Figure 1. Gingival stromal stem cells (G-MSSCs) showed mesenchymal stem cell properties. i) CD44, CD29, CD106, CD105, CD146, and CD90 are mesenchymal stem cell surface receptors that were detected on G-MSSCs, except CD106. CD106 is a subpopulation of mesenchymal stem cells that addresses immunomodulatory properties. Hematopoietic stem cell surface receptors CD14, CD34, CD45; CD3, HLA DR were found to be negative. Expression of CD73 as a cluster of differentiation and HLA-ABC as an evidence of inflammatory condition was found to be positive ii) G-MSSCs have low adipogenic differentiation potential, whereas osteogenic differentiation was strong (a) Adipogenic differentiation: The black arrows show positively stained G-MSSCs for lipid vesicules with Oil Red O stain. (b) Osteogenic differentiation: The calcium granules were stained black with Alizarin Red stain in the osteogenic differentiation medium on the G-MSSCs (40x, Olympus CKX41, Tokyo, Japan)
Figure 2. i. L. rhamnosus ATCC9595 modulated…
Figure 2. i. L. rhamnosus ATCC9595 modulated CXCL8 (a) and IL-10 (b) secretion. (a) The gingival stromal stem cells (G-MSSCs) secreted an amount of CXCL8 without any stimulation. L. rhamnosus and P. gingivalis reduced CXCL8, as known. The reduction was found to be significant according to Mann-Whitney U test (p<0.05). A L. rhamnosus and P. gingivalis coculture exhibited a stimulant effect for CXCL8 as an inflammatory inducer, according to a Kruskal-Wallis test, which verified our hypothesis (p<0.05). IFN provoked GMSSCs to secrete CXCL8 (p<0.05). On the other hand, CXCL8 was found to be reduced on L. rhamnosus pretreated G-MSSCs when induced with IFN (p<0.05). (b) In contrast to CXCL8, the G-MSSCs did not secrete IL-10. L. rhamnosus and P. gingivalis increased IL-10 secretion, compared with G-MSSCs (p<0.05). The L. rhamnosus and P. gingivalis coculture reduced IL-10, since CXCL8 was increased (p<0.05). IL-10 was reduced on IFN-induced G-MSSCs, while CXCL8 was increased. L. rhamnosus -pretreated G-MSSCs induced IL-10 in IFN stimulation (p<0.05). ii . TLR expression was found to be synchronized with CXCL8 and IL-10 secretion. (a) G-MSSCs did not express TLR2 or 4 (<99.3%) b) G-MSSCs expressed TLR4 (42.1%) when stimulated with IFN (p<0.01). c) G-MSSCs pretreated with L. rhamnosus decreased the expression of TLR4 (24%) in IFN-induced inflammatory conditions (p<0.01). d) P. gingivalis -induced TLR4 expression (11.1%; p<0.01). On the other hand, both TLR4 and TLR2 were expressed (1.1%). Decreased CXCL8 represents a gingipain effect, since we expected increased CXCL8 due to expressed TLR4 e) The L. rhamnosus and P. gingivalis coculture was able to reduce TLR4 expression to 0.5% (p<0.01). On the other hand, TLR2 was found to be 1.7% (p<0.01), which indicates the TLR2- dependent CXCL8 secretion. f) L. rhamnosus, when used alone, could induce TLR4 (4.5%) (p<0.01). G-MSSCs: Gingival mesenchymal stromal stem cell. IFN: Interpheron –γ. * indicates statistically significant groups

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