Lactobacillus rhamnosus could inhibit Porphyromonas gingivalis derived CXCL8 attenuation

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.


INTRODUCTION
The gram-negative, anaerobic bacterium Porphyromonas gingivalis is considered to be one of the key pathogens in periodontitis 20, 24 . P. gingivalis possesses a number of pathogenic properties that lipopoylsaccharides, and gingipains 24 . Accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. P. gingivalis stimulates an innate immune response and induces the expression of inflammatory mediators, but it can downregulate the host immune response at the same time. In other words, P. gingivalis has evolved various mechanisms to escape host immune systems by invading host cells and disrupting signaling pathways through cytokine and receptor degrading 18 . disease resulting from a complex polymicrobial infection in which the disruption of the homeostasis between the subgingival microbiota and the host defense leads to the destruction of the tooth-supporting tissue 25 . As a result of bacterial encounters, the host cells synthesize and release cells to the site of infection 19, 24 . CXCL8 is an important chemokine that attracts neutrophils to the site of infection. The CXCL8 chemokine is expressed and produced by different cell types keratinocytes, epithelial cells, and lymphocytes 10 . P. gingivalis has a different way of interacting with the host's innate immune response, compared to other pathogenic, gram-negative bacteria, which is recognized as inhibiting CXCL8 expression. The attenuation of CXCL8 may delay the defense mechanisms of the host and allow P. gingivalis to escape the immune system, thus creating more damage to the surrounding tissue 17 .
The ability of the immune system of the host to sense, recognize, and respond to periodontal associated pathogens is an important determinant in the pathogenesis of periodontitis. This ability is largely mediated by the innate immune system via the expression of toll-like receptors (TLRs) 16 .
3, 4, and 5, and ligands binding to these receptors leads to the secretion of CXCL8 21 . Moreover, TLR2, which recognizes gram-positive bacterial cell walls, P. gingivalis 12 . Studies have shown that P. gingivalis could signal via TLR2, TLR4, or both 29 .
Conventional periodontal treatment is often not sufficient by itself to control destructive diseases 1 . This requires the development of novel and effective therapeutic strategies that are adjunctive to clinical periodontal treatment. The use of probiotics is one of the several approaches being considered for the treatment of periodontitis 3 . Probiotic therapy has recently gained massive effects on general and oral health as well as for being an important complement to antibiotic treatment. Furthermore, the administration is simple, inexpensive, and safe 23 . Animal and human studies have shown that the use of probiotics is emerging as a potential adjunctive therapy for periodontitis, although the underlying mechanisms 22 . Our team has supported conducting in vitro studies before using probiotics in clinical trials. In this study, the hypothesis tested was that CXCL8 suppression by P. gingivalis could be prevented by the probiotic strain L. rhamnosus ATCC 9595 through co-aggregation, competitive adhesion, and the expression of TLRs.

Isolation and identification of gingival mesenchymal stromal stem cells
Gingiva samples were obtained from four healthy subjects aged 21 to 24 years old during wisdom-tooth extraction. The experimental protocol was reviewed and approved by the Institutional Ethical Board, and informed consent form was obtained from each subject. Gingival mesenchymal stromal according to Zhang, et al. 30 (2009). Differentiation protocol was conducted according to the protocol described as Pittenger, et al. 26 (1999).

Bacteria and growth conditions
L. rhamnosus ATCC9595, the probiotic strain, and P. gingivalis ATCC33277 were obtained from American Type Culture Collection. The probiotic strain was cultured in MRS broth (de Man, Rogosa, Sharpe, MERCK) under aerobic conditions at 37°C for 18 h. P. gingivalis was cultured under anaerobic conditions (80% N 2 , and 10% H 2 ) at 37°C in an anaerobic chamber (Electotek Anaerobic workstation, UK) and maintained on Schaedlar -1 ), 5% ml -1 ). The number of bacteria at the beginning of the experiment was adjusted to McFarland 2 (~1×10 8 ) and expressed as cfu mL -1 by the serial dilution technique.

Auto-and co-aggregation assays
The auto-aggregation assay was performed according to the methods developed by Del Re, et al. 9 bacteria were grown for 18 h at 37°C with the appropriate growth medium. The culture was harvested by centrifugation at 5000 g for 15 min, washed twice, and resuspended in phosphatebuffered saline (PBS) to give viable counts of approximately 10 8 cfu mL -1 . Cell suspensions (4 mL) were mixed by vortexing for 10 s, and auto-aggregation was determined during 4 h of incubation at room temperature. At the end of the incubation period, 0.1 mL of the upper suspension was transferred to another tube with 0.9 mL of PBS, and the absorbance (A) was measured at 600 nm. The auto-aggregation percentage was expressed as (1-(A t /A 0 ))×100, where A t represents the absorbance at the end of incubation time and A 0 the absorbance at t=0.
The method for preparing cell suspensons for co-aggregation was the same as that for the autoaggregation assay. Equal volumes (2 mL) of each cell suspension were mixed together in pairs by vortexing for 10 s. Control tubes containing 4 mL of each bacterial suspension on its own were set up at the same time. The absorbance (A) of the suspensions at 600 nm was measured after mixing and after 4 h of incubation at room temperature. Samples were taken in the same way as in the auto-aggregation assay. The percentage of coaggregation was calculated using the equation given

Competitive adhesion of bacterial strains to G-MSSCs
For the competitive adherence assay, a total cell number of 200 000 G-MSSCs per well were seeded on glass coverslips and incubated for 24 h in 6 well plates. Before the experiment, the culture medium was changed with antibiotic-free medium. G-MSSCs were challenged with bacteria at a multiplicity of infection (MOI) of 1:100 (10 8 bacteria well -1 ) at 37°C in 5% CO 2 for 1 h. L. rhamnosus and P. gingivalis were provided equal chances to bind at the same ratio and at the same time 8 . At the end of 1 h, the light microscopy, as described by Bernet, et al. 2 (1993). For each monolayer on a glass coverslip, the numbers of adherent bacteria on 100 different, randomly selected cells were evaluated.

Effect of L. rhamnosus on G-MSSCs upon P. gingivalis stimulation
G-MSSCs were allowed to attach to and grow on 24-well tissue culture plates (50 000 cells well -1 ) containing culture medium (Costar, Corning, USA). After 24 h of incubation, the medium was discarded, washed with PBS, and replaced with new medium without Pen/Strep. cells that were pretreated with L. rhamnosus (MOI 1:100) for 12 h at 37°C and 5% CO 2 . Subsequently, the culture wells were washed (Invitrogen, USA) or P. gingivalis (MOI: 1:100) for another 12 h. At the end of the experiment, culture supernatants were harvested to determine their cytokine levels, and the cells were prepared

Determination of cytokine production
The co-culture supernatants were collected and centrifuged at 4000 g for 3 min at 4°C. The amounts of CXCL8 and IL-10 secreted into the medium during the co-culturing with the bacteria were measured by using the Human Ultrasensitive ELISA Kit (Invitrogen, USA). The culture supernatants were diluted according to the manufacturer's instructions. The ELISA limits were 0-25 pg mL -1 for CXCL8 and 0-50 pg mL -1 for IL-10.
G-MSSCs were prepared by treatment with Trypsin/EDTA (Invitrogen, USA), transferred to centrifuged (200 g × 5 min), and washed with PBS. The cells were fixed by adding 2 mL of FACS lysing solution (BD Bioscience, Heidelberg, for 10 min at room temperature in dark. G-MSSCs were centrifuged (200 g × 5 min) and washed with 2 mL of wash buffer (1% BSA, 0.1 % NaN 3 PBS). Then, the cells were stained for expression of TLR-2 and TLR-4 on G-MSSCs with anti-TLR-2 FITC and anti-TLR-4 PE antibodies (eBioscience, USA). The harvested cells were incubated within the isotype IgG, after which they were used as controls. Appropriate isotype controls (IgG-2a) were also used in each case. Positively stained cells were system (CEllQuest ProSoftware, BD Biosciences, Heidelberg, Germany). The experiments were done in triplicate; representative histograms are presented in the Results section.

Data analysis
Aggregation, adhesion, ELISA, and flow cytometer assays were carried out in two individual experiments involving 3 replicates. Auto-and co-aggregation was analyzed by Spearman's rho test, whereas Mann-Whitney U test was applied for comparing adhesion and competitive adhesion assays. CXCL8 and IL-10 determination from culture supernatants were analyzed by comparing the G-MSSCs with bacteria treated cells and IFN induced groups using Mann-Whitney U and Kruskal-Wallis test. TLR expression on cell surfaces by IFN-and/or P. gingivalis ATCC33277 and ability of the probiotic strain to reduce TLR-4 expression were investigated by comparing two proportions by Z-test. A value of p<0.01 and p<0.05 was considered to be

RESULTS
The expansion of G-MSSCs in culture was successful for at least eight passages in all of the samples. The common MSC markers CD106, CD105, CD73, CD29, CD90, CD146, and CD44 and the hematopoietic markers, CD3, CD45, CD14, HLADR, HLA-ABC, and CD34, were tested, suggesting a mesenchymal origin for the cells (Figure 1-i).

G-MSSCs show adipogenic and osteogenic differentiation capacity, but potency is low
Around day 10 (range of 8-14) of exposure to adipogenic medium, very small lipid droplets were barely visible by inverted microscope within the bulk of the cells obtained from gingival tissues (Figure 1-ii.a). Oil Red O staining was performed on the culture in adipogenic medium on day 30.
The morphology and staining characteristics of the differentiating cells were consistent with a preadipocyte phenotype. Small, dark deposits were visible on the culture plates of G-MSSCs after 7-10 days, and they increased in the following days within osteogenic differentiation culture. Amorphous deposits were present on the plates by positive Alizarin Red S staining at day 30 of culture, indicating the in vitro osteogenic potential of the cells (Figure 1-ii.b).

L. rhamnosus could co-aggregate with P. gingivalis
The results showed that the strains exhibited a strong auto-aggregation phenotype ( Table 1). The L. rhamnosus and P. gingivalis co-aggregation according to Spearman's rho test **± refers to the standard deviation

L. rhamnosus inhibits the adhesion of P. gingivalis
The morphology of G-MSSCs following treatment with a viable probiotic strain and P. gingivalis was examined by light microscopy. No obvious morphological changes induced by the bacteria were observed. We distinguished the attached P. gingivalis from the probiotic strain according to their Gram stain properties. We also determined the adhesion of the strains separately; L. rhamnosus showed higher adhesive properties than P. gingivalis on G-MSSCs (p<0.01; Table 2). L. rhamnosus and P. gingivalis 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 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

DISCUSSION
This in vitro study aimed to investigate whether the probiotic strain L. rhamnosus ATCC 9595 could prevent the P. gingivalis welded CXCL8 supression through co-aggregation, competitive adhesion, and the expression of TLRs. We chose to use the probiotic L. rhamnosus because the strain was reported to induce IL-10 secretion and produce high levels of exopolysaccharides (EPSs) in different works 4,6 . EPSs are one of the primary metabolic products of lactic acid bacteria, and have received an increasing amount of attention because of their 22 . The ability of a microorganism to surround itself with a highly hydrated EPS layer may provide it with protection against desiccation and predation. We suggest that in terms of oral environments in clinical studies, considering the stressful conditions created by saliva and tooth surfaces, high EPS-productive strains of L. rhamnosus may survive better.
Although the mechanisms of action are not fully understood, it is generally accepted that the ability of probiotics to co-aggregate with pathogens is a desired property. Co-aggregation has an important ecological role as an integral process in the development and maintenance of mixed-species biofilm communities, especially in oral cavity. bacteria need to achieve an adequate mass through aggregation. We used a simple and robust spectrophotometric method that has been shown to correspond well to more sophisticated radioactive L. rhamnosus could co-aggregate with P. gingivalis after 4 h. The co-aggregation ability of the probiotic strain could enable the formation of a barrier that prevents colonization by pathogenic bacteria 9 .
In addition to aggregation, attachment is an and in most cases, aggregation ability is related to cell adherence properties 9 . We examined the competitive adhesion of the probiotic strain and P. gingivalis on G-MSSC monolayers that were grown study investigating L. rhamnosus and P. gingivalis's competitive adhesion on G-MSSCs. L. rhamnosus  which is in good agreement with studies conducted with Caco-2 cells 11,27 . It is feasible to take advantage of L. rhamnosus, which could confer advantage to this strain a competitive in vivo. It is well known that P. gingivalis adheres to and invades epithelial in vitro. The invasion of epithelial a mechanism applied by bacteria to evade the immune system of the host 1 . It was reported that invasion could occur after 6 h of incubation 25 . Based on these reports, we incubated the probiotic strain and P. gingivalis for 1 h to prevent the invasion into the GMSSCs. We showed that the probiotic strain inhibited the adhesion of P. gingivalis, although the mechanism remains unclear. Our results do not explain whether the exclusion of P. gingivalis of bacteria prevented access to the cell surfaces of organisms. These results show a potential for adhesive and co-aggregative L. rhamnosus to inhibit the cell association and cell entry of P. gingivalis.
Many studies have tried to induce a microbiological shift or a clinical probiotic effect in an already matured oral microbiological environment. However, effects. We suggest that L. rhamnosus with its coaggregation ability and strong adhesive properties, may survive better. As a consequence of adhesion, the preference would orient the cell response according to the probiotic strain, since probiotics can also activate and modulate the immune system 7 . In the present study, we demonstrated that probiotic P. gingivalis interactions can inhibit CXCL8 attenuation. It is known that secreted CXCL8 proteins are downregulated when cells are challenged with P. gingivalis 14 . The suggested mechanism comprises the downregulation of CXCL8-mRNA and/or the degradation of CXCL8 by proteases (called gingipains). Strong evidence has shown that using antibiotics or deleting one of the protease genes (rgpA, rgpB, or kgp) in P. gingivalis did not dramatically affect CXCL8 attenuation 14 . We showed that 12 h of exposure to viable P. gingivalis suppressed CXCL8 production in the cells, which is inconsistent with literature 17, 25 . On the other hand, when the cells were pretreated with the probiotic strain before P. gingivalis stimulation, the CXCL8 levels were found to have increased, indicating that P. gingivalis proteases might be degraded by L. rhamnosus or P. gingivalis-probiotic strain co-aggregation, which may activate or deactivate any structures on the bacteria cell wall responsible for the degradation of CXCL8. Indeed, we need to demonstrate the molecular mechanism in this interaction. We screened the effects of bacteria on cytokine secretions when used alone. Low levels of CXCL8 and IL-10 were determined in the G-MSSCs culture supernatant, as corroborated by Tonetti, et al. 28 (1994), who reported that low-level expression of CXCL8 in healthy tissue most likely contributes to the remarkable ability of the host to limit periodontal bacterial growth. To further investigate the effect of probiotics on CXCL8 levels were downregulated and IL-10 levels were found to have increased. Such immunomodulation induced by probiotics is important for maintaining the host-microbe homeostasis without triggering Because TLR activation plays a vital role in cytokine production, we measured the expression of TLRs after treatment with the probiotic strain and P. gingivalis. It must be noted that P. gingivalis lipopolysaccharide (LPS), another putative virulence factor, is suggested to evade recognition by the host via TLR-4 29 . The lack of to P. gingivalis is counterintuitive, since that this is a gram negative bacterium that expresses a LPS. However, P gingivalis 4-phosphatases and a deacylase, which, in concert, generate a tetracylated and dephosphorylated lipid, a structure that is biologically inert 7 . In contrast to this report, in our study we observed TLR-4 expression (11.1%), but reduced CXCL8 levels and TLR-2. This result could be explained by the degradation of secreted CXCL8 by gingipains. Moreover, P. gingivalis's effect was clearly altered when L. rhamnosus was added to the culture. CXCL8 and TLR-2 were found to be enhanced, while TLR-4 expression was reduced. Several groups have found that TLR-2 is required for a full cytokine response to infection with P. gingivalis, raising the question of whether P. gingivalis evades clearance by reducing recognition through TLR-2, rather than TLR-4 5,13 . Our results clearly showed that L. rhamnosus together with P. gingivalis boosts CXCL8 production while enhancing TLR 2 and inhibiting TLR-4 expression. Previous reports showed TLR-2, rather than TLR-4, to be critical for the host response to infection with P. gingivalis 5,13 . Gingipain inhibition by L. rhamnosus and its effect on TLR-2 expression should be determined to conclusively show that L. rhamnosus inhibits CXCL8 attenuation through an enzyme or polysaccharides.

CONCLUSION
The immunomodulatory probiotic strain L. rhamnosus ATCC 9595 is proposed to be essential for maintaining healthy tissue, with multiple roles including co-aggregation, adhesion, and priming defense against P. gingivalis ATCC 33277. The default state of oral tissues, such as in the gut, is probiotics that modulate TLR signaling. There evaluated the effects of probiotics, possibly because they used different doses, treatment durations, bacterial species, and application forms. These screenings to select appropriate probiotic strains for preventing gingivitis or periodontitis and other oral health diseases. Thus, new in vitro studies before they are introduced into clinical practice for periodontal therapy. The results of the present L. rhamnosus ATCC 9595 to prevent P. gingivalis induced inflammation and CXCL8 attenuation. However, our results should be proven in vivo, through both human and animal trials.