Lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli: differential and interactive effects on novelty-induced hyperlocomotion, blood cytokine levels and TRL4-related processes

Lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, activates Toll-like receptors (TLRs). Porphyromonas gingivalis (Pg) may be involved in the progression of periodontal disease. Mice exposed to a novel environment show hyperlocomotion that is inhibited by systemic administration of LPS derived from Escherichia coli (Ec-LPS). However, whether Pg-LPS influences novelty-induced locomotion is unknown. Accordingly, we carried out an open field test to analyse the effects of Pg-LPS. For comparison, effects of Ec-LPS were also studied. We also investigated the influence of systemic administration of Pg-LPS or Ec-LPS on IL-6, TNF-alpha, and IL-10 levels in blood, as they could be involved in the changes in locomotion. The TLR4 receptor antagonist TAK-242 was used to study the involvement of TLR4. Since Pg-LPS may block TLR4 in vitro, we analysed the effects of Pg-LPS on Ec-LPS-induced changes in behavioural and biochemical parameters. Male ddY mice were used. Compounds were administered intraperitoneally. Ec-LPS (840 µg/kg), but not Pg-LPS (100, 500 and 840 µg/kg), inhibited novelty-induced locomotion, which was reversed by TAK-242 (3.0 mg/kg). Ec-LPS (840 µg/kg) increased blood levels of IL-6 and IL-10, which was antagonized by TAK-242 (3.0 mg/kg). However, TAK-242 did not inhibit Ec-LPS-induced increases in TNF-alpha levels in blood. Pg-LPS (100, 500, and 840 µg/kg) did not alter blood IL-6, TNF-alpha, or IL-10 levels. The Ec-LPS-induced increase in blood IL-10, but not IL-6 and TNF-alpha, levels was inhibited by Pg-LPS (500 µg/kg). These results suggest that TLR4 stimulation mediates the inhibition of novel environment-induced locomotion in mice following systemic administration of Ec-LPS, while also increasing blood IL-6 and IL-10 levels. In contrast, Pg-LPS did not exhibit these effects. The present study also provides in vivo evidence that Pg-LPS can inhibit TLR4-mediated increases in blood IL-10 levels, which is thought to prevent the development of periodontal disease.


Introduction
Porphyromonas gingivalis (Pg) is a Gram-negative, rod-shaped bacterium that may be involved in the progression of periodontal disease.Lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall derived from Pg (Pg-LPS), has been implicated as a key molecule in the development of periodontitis.
In vitro studies using cultured cells from periodontal tissue have revealed Pg-LPS to induce effects distinct from LPS derived from the prototype Gramnegative, rod-shaped bacterium Escherichia coli (Ec-LPS).Notably, increases in interleukin (IL)-6 expression observed in gingival fibroblasts [1] and periodontal ligament cells [2] following treatment with Pg-LPS were reduced relative to those following treatment with Ec-LPS.Additionally, treatment with Pg-LPS failed to alter IL-6 and tumor necrosis factor (TNF)-alpha levels significantly in dental pulp stem cells, while Ec-LPS treatment markedly increased these cytokines [3].Moreover, treatments with Pg-LPS had no significant effect on cytokine production in monocyte-derived dendritic cells, whereas Ec-LPS treatment led to a significant increase in IL-6 and IL-10 production [4].
Despite these observed differences in cytokine responses, the specific mechanisms underlying the divergent effects of Pg-LPS and Ec-LPS on cytokine production remain unclear.Nevertheless, it is plausible that variations in receptors interacting with Pg-LPS and Ec-LPS may contribute to these discrepancies.Toll-like receptors (TLRs) are well-known pattern recognition sites that bind pathogenassociated molecules, ultimately activating immunity and host defense [5].Notably, LPS stimulates a subtype of TLR known as TLR4, which is expressed on the plasma membrane and mediates various processes, including cytokine synthesis and secretion.Studies using cultured monocytic cells have suggested that increases in IL-6 and TNF-alpha levels induced by Ec-LPS are mediated via TLR4 activation.On the other hand, the effects of Pg-LPS on IL-6 and TNF-alpha were proposed to be mediated via TLR4 and/or another subtype of TLR known as TLR2 [6].Moreover, differences in the effects of Pg-LPS and Ec-LPS can be attributed to their differential abilities to activate TLR4; previous research has shown that Ec-LPS strongly stimulates TLR4, whereas Pg-LPS weakly stimulates [7][8][9][10] or blocks [11][12][13][14] TLR4.
In vivo studies in rats have demonstrated that Pg-LPS induces effects on extracellular cytokine levels that differ from those induced by Ec-LPS.For example, our studies using urethane-anesthetized rats indicated that intra-gingival administration of Ec-LPS failed to alter cytokine levels, while Pg-LPS increased gingival extracellular levels of TNF-alpha [15].Systemic administration of Ec-LPS in mice is known to induce a variety of sickness behaviors, such as reductions in water and food intake and loss of body weight [16].These behavioral changes are suggested to be mediated by increases in the synthesis and release of cytokines that can be detected in the systemic circulation [16].Notably, the sickness behavior induced by systemic administration of Ec-LPS also includes reduction in locomotor activity [17].However, it remains unknown whether systemic administration of Pg-LPS in mice produces inhibitory effects on locomotion similar to those induced by Ec-LPS.Therefore, we conducted an open field test to examine whether a single systemic administration of Pg-LPS in mice affects locomotor activity in a novel environment, to further characterize the effects of Pg-LPS in vivo.For comparison, we also analyzed the effects of Ec-LPS on novelty-induced increases in locomotor activity under the same experimental conditions.Additionally, we investigated the influence of systemic administration of Pg-LPS or Ec-LPS on blood levels of cytokines, particularly IL-6, TNF-alpha and IL-10, as these cytokines may have pro-inflammatory (IL-6 and TNF-alpha) or antiinflammatory (IL-10) effects that could be involved in the observed changes in mouse behavior.Furthermore, animal experiments have shown that systemic inflammation induces increases in spleen mass [18].For example, repeated oral administration of Pg has been found to increase spleen weight in mice [19].The spleen plays a crucial role in modulating immune responses by promoting the differentiation and activation of T and B cells.Thus, we investigated the influence of systemic administration of Pg-LPS or Ec-LPS on spleen weight, together with the numbers of T cells and B cells using flow cytometry.To elucidate the involvement of TLR4 in the effects of LPS on behavioral and biochemical parameters in mice, we co-administered TAK-242, a selective TLR4 receptor antagonist [20].Given the in vitro experiments suggesting that Pg-LPS may exhibit partial agonist-or antagonist-like properties on TLR4, as discussed above [7][8][9][10][11][12][13][14], we proceeded to analyse the effects of Pg-LPS on Ec-LPSinduced changes in behavioural and biochemical parameters in mice.
Preliminary results from these investigations have been presented at the Annual Meeting of the Japanese Pharmacological Society, 2022, and the Annual Meeting of the Japanese Association for Oral Biology, 2023.

Animals
Male ddY mice (Sankyo Laboratory Service Co. Ltd., Tokyo, Japan), weighing between 25 and 30 g were used.These were kept at constant room temperature (23 ± 2 ºC) and relative humidity (55 ± 5%) under a 12 h day:night cycle (light on: 07.00 a.m.), with ad libitum access to food and water.All experiments were approved by the Animal Experimentation Committee of Nihon University School of Dentistry at Matsudo (AP19MAS015) and were performed in accordance with national and international guidelines for the care and welfare of animals.All efforts were made to minimise animal suffering and to reduce the number of animals used.

Drugs and treatments
Commercially available LPS derived from Ec and Pg, namely Ec-LPS (O55:B5; Sigma-Aldrich, St. Louis, MO, USA) and Pg-LPS (LPS-PG Ultrapure; InvivoGen, San Diego, CA, USA) were used.They were diluted with saline and aliquots were stored at -20℃ prior to use within two months.TAK-242 (Sigma-Aldrich) was used as a TLR4 antagonist.It was diluted with saline and a small amount of dimethyl sulfoxide (<0.1%) was added immediately prior to use.The doses of these compounds were determined by a series of pilot experiments based on previous behavioural studies (LPS: [17,21]; TAK-242: [20]).
Compounds were injected i.p. in a volume of 0.1 ml per 10 g body weight, with each mouse used only once.Mice were treated with LPS or its corresponding vehicle 4 h prior to the open field test and the subsequent measurements of spleen weight, flow cytometry, and blood cytokine levels.TAK-242 or its vehicle was administered 1 h before designated procedures.

Open field test
On the day of experiments, each mouse was individually placed in a plastic cage (length × width × height: 32 × 21 × 13 cm), the floor of which was covered with sawdust to a depth of approximately 2 cm, and habituated to these conditions for at least 60 min.Then, mice treated with LPS and/or TAK-242 or their corresponding vehicles (see Drugs and treatments) was transferred to a Plexiglas box (length × width × height: 30 × 30 × 20 cm) that was slightly larger and lacked floor sawdust.
Locomotion in the novel environment was recorded for a period of 30 min via a video camera located above the Plexiglas box and total distance traveled (cm) was determined by an automated system for behavioural assessment (SMART, Panlab, Barcelona, Spain).In designated experiments we carried out additional analyses of locomotion in the center (20 × 20 cm) vs the periphery (within 5 cm of each wall) of the Plexiglas box, since avoiding the center zone of the novel environment is regarded as a sign of anxiety that could be elicited by systemic administration of LPS.After evaluation of each mouse the Plexiglas box was cleaned with ethanol followed by water before proceeding to the next mouse.

Measurement of spleen weight and flow cytometry
After treatments with LPS and/or TAK-242 or their corresponding vehicles (see Drugs and treatments) mice were euthanized by isoflurane (5%) inhalation.Their spleens were collected and weighed by a precision electronic weighing scale and single-cell suspensions were prepared in saline using a homogenizer.Surface markers of splenocytes were identified using monoclonal antibodies in conjunction with two immunofluorescence analysis with a flow cytometer (BD Accuri C6 Plus: BD, Mountain View, CA, USA).Antibodies used were Fluoroscein isothiocyanate (FITC)-conjugated anti-mouse CD4 and CD21/CD35 (CR2/CR1: Biolegend, San Diego, CA, USA) and Phycoerythrin (PE)-conjugated anti-mouse CD3 (17A2: Proteintech, Rosemont, IL, USA).Isotype controls used were FITClabelled Rat IgG2b Isotype Control (LTF-2) (Proteintech, Rosemont, IL, USA) and PE-labelled Rat IgG2b Isotype Control (Becton Dickinson Pharmingen, San Jose, CA, USA).Double-labelled surface phenotypes were CD4/CD3 and CD3/CD21.Cells were pre-incubated with anti-CD16/CD32 antibody to block nonspecific antibody binding.Then, cells were incubated with the above specific antibodies for 30 min and washed twice in 1% bovine serum albumin in phosphate buffered saline.
Seven-amino-actinomycin D (Biolegend, San Diego, CA, USA) was added before analysis with a flow cytometer to exclude nonviable cells.Data were analyzed using commercial software (FlowJo, BD Biosciences, Franklin Lakes, NJ, USA).

Measurement of blood cytokine levels
A disposable lancet (4 mm, Goldenrold Animal Lancet, MEDIpoint, Mineola NY, USA) was used to take blood samples from the submandibular vein of mice treated with LPS and/or TAK-242 or their corresponding vehicle (see Drugs and treatments).Approximately 0.5 ml of blood was quickly collected without total anesthesia [22].Blood was allowed to coagulate for 10-15 min at room temperature, then centrifuged at 1900 × g for 10 min at 4℃, with separated sera then stored at -70℃ until further analysis.Levels of IL-6, TNF-alpha and IL-10 in serum were determined using beadbased Multi-Plex kits (MILLIPLEX ® Mouse Cytokine/Chemokine Magnetic Bead Panel 96-Well Plate Assay, Merck KGaA, Cat.No. MCYTOMAG-70K-03, Rockland, MA, USA).In order to remove particulates, samples were centrifuged at 16000 × g for 4 min at 4℃ before assay and diluted 2-fold using medium provided by the kit manufacture.Twenty-five µl of diluted samples were then transferred into a 96-well plate and incubated overnight at 4℃ with shaking to immobilize antibody beads.Liquid was removed, followed by two washes, and detection beads were then added into each well.The plate was then incubated for 1h at room temperature, followed by addition of streptavidin-phycoverthrin for 30 min with shaking.After the supernatant was removed, 150 µl of drive fluid to resuspend the beads was added to each well and the plate was read using MAGPIX plate reader with xPOINT software (Luminex® 100/200™ System, Luminex Corp., Austin, TX, USA).Data were analyzed using MILLIPLEX Analyst software.

Statistical analysis
All values are expressed as mean and S.E.M. Comparisons of (1) effects of various doses of Pg-and Ec-LPS on distance travelled and (2) effects of TAK-242 or Pg-LPS on Ec-LPS-induced changes in distance travelled, body weight, spleen weight, cellular components of spleen and blood cytokine levels were performed using one-way ANOVA followed by post hoc Scheffé's test where appropriate.
Effects of Ec-LPS on distance travelled in central vs peripheral zones of the novel environment were compared using Student's t-test.Statistical significance was considered to be P < 0.05.

Ec-LPS-induced inhibition of novelty-induced locomotion is evident in both the central and peripheral zones
As differential effects on locomotion in the peripheral vs central zones of the open field implicate anxiety-/depression-related processes [23], we analyzed the effects of Ec-LPS (840 µg/kg) on locomotion in these two zones.Ec-LPS treatment inhibited locomotion in both the central and peripheral zones (Fig.
Though there appeared to be some numerical variation, neither body nor spleen weight differed significantly across these groups (Table 1).Next, we assessed the cellular composition of the spleen in these same experimental groups by flow cytometry and determined the numbers of CD4 + helper T cell and CD21 + mature B cell subsets.Though there appeared to be some numerical variation, neither the number of CD4 + T cells nor the number of CD21 + B cells differed significantly across these groups (Table 2).

Discussion
The present study indicates that systemic administration of Ec-LPS inhibits novel environment-induced increases in locomotor activity by activating TLR4, as this effect was inhibited by the selective TLR4 antagonist TAK-242.To further clarify this Ec-LPS-induced inhibition of locomotor activity, more detailed analysis indicated this inhibition to be evident similarly in both the peripheral and central zones of the open field.As anxiety-and/or depression-related processes are reflected in differential effects between these zones [23], such processes are not implicated in the present findings.Rather, they may reflect evidence that LPS-induced reductions in locomotor activity, including spatial exploration, relate to prevention of the spread of inflammation and promotion of healing [16].
In contrast to Ec-LPS, systemic administration of Pg-LPS did not influence novel environment-induced increases in locomotor activity.Previous findings from our intra-gingival injection study in urethane-anesthetized rats showed that Pg-LPS, but not Ec-LPS, produced a transient increase in extracellular TNF-alpha levels at the injection site [15].The present findings provide further evidence that Pg-LPS can elicit effects that differ from those of Ec-LPS not only in in vitro but also in vivo.
Several in vitro studies have indicated that TLR4 can be weakly stimulated [7][8][9][10] or inhibited [11][12][13][14] by Pg-LPS.Based on these findings, it is plausible that the present TLR4-mediated decrease in locomotion induced by Ec-LPS might be counteracted by systemic administration of Pg-LPS.However, it should be noted that while coadministration of Pg-LPS with Ec-LPS tended to inhibit Ec-LPS-induced changes in locomotion, this failed to attain statistical significance.
It is conceivable that systemic administration of LPS may potentially promote the differentiation of T cells and B cells.This is based on the belief that T cells interact with antigen-presenting cells, including dendritic cells and macrophages that recognize LPS, resulting in their activation and differentiation into specific subtypes, such as helper T cells.Additionally, LPS activates B cells, leading to their differentiation into B cells involved in antibody production, thereby enhancing their ability to produce antibodies.Moreover, systemic administration of LPS is known to induce systemic inflammation and increase spleen weight in experimental animals [18].However, contrary to these assumptions, the LPS treatments in this study did not increase the number of T or B cells derived from mouse spleen, nor did they increase spleen weight.These results indicate that systemic administration of not only Pg-LPS but also Ec-LPS could not alter the compositions of lymphocytes, at least within the experimental period of the present studies.Furthermore, the lack of significant changes in spleen weight suggests that these LPS treatments are unlikely to induce material systemic inflammation within this experimental period.
In agreement with an earlier report suggesting that an increase in cytokine synthesis and release mediates the sickness behavior induced by systemic administration of LPS, including decreased novelty-induced locomotion in experimental animals [16], we observed marked increases in blood levels of IL-6, TNF-alpha, and IL-10 in mice treated with Ec-LPS.Interestingly, the biological processes involved in the increases in levels of TNF-alpha were not the same as those for IL-6 and IL-10 under the present experimental conditions.Notably, the TLR4 antagonist TAK-242 inhibited Ec-LPS-induced increases in blood IL-6 and IL-10 levels but had no effect on TNF-alpha levels.This suggests that IL-6 and IL-10 are likely increased by TAK-242-sensitive TLR4 activation.
Underlying reasons for the failure of TAK-242 to affect Ec-LPS-induced increases in TNF-alpha levels are not yet known, but several possibilities may be considered.Firstly, the contribution of intra-cellular TLR4, which may be less accessible to compounds such as TAK-242, could explain its inactivity on TNF-alpha levels [24].Secondly, differences in TAK-242 concentrations at TLR4 may result in differential blockade, influencing IL-6 and IL-10 levels differently from those of TNF-alpha.For example, under the present in vivo conditions TAK-242 might attain a higher concentration at TLR4 sites involved in IL-6 and IL-10 synthesis and release, while attaining a lower concentration at TLR4 sites involved in TNF-alpha synthesis and release.Further investigation would be necessary to clarify these possibilities and gain deeper insights into the mechanisms underlying the differential effects of TAK-242 on cytokine levels.
In contrast to Ec-LPS, systemic administration of Pg-LPS did not influence novelty-induced locomotion in mice and did not alter basal levels of IL-6, TNF-alpha or IL-10 in blood.These results align with previous in vitro findings using gingival fibroblasts, which showed that increases in IL-6 expression induced by Pg-LPS were smaller than those induced by Ec-LPS [1].The present results provide in vivo biochemical evidence for clear differences in the influences of Pg-LPS and Ec-LPS on blood IL-6, TNF-alpha and IL-10 levels when administered systemically.Since in vitro studies have shown that Pg-LPS weakly stimulates [7][8][9][10] or inhibits [11][12][13][14] TLR4, we investigated the effects of co-administration of Pg-LPS on Ec-LPSinduced increases in blood IL-6, TNF-alpha and IL-10 levels that appear mediated by TLR4 activation.Distinct from Ec-LPS-induced increases in blood TNF-alpha levels, which were not inhibited by TAK-242, Ec-LPS-induced increases in blood IL-10, but not IL-6, were suppressed by co-administration of Pg-LPS.The reduction in noveltyinduced locomotor activity of mice induced by Ec-LPS is likely mediated by increases in blood IL-6, as opposed to TNF-alpha and IL-10.This conclusion is their potential contribution to the development of Alzheimer's disease [29].Similarly, the impact on cardiovascular cells is noteworthy, with possible implications for atherosclerosis [30].

Conclusion
In summary, this study provides compelling in vivo evidence that Ec-LPS and Pg-LPS exert differential effects on both locomotor activity and cytokine responses in blood.The systemic administration of Ec-LPS inhibits novel environment-induced locomotor activity in mice through TLR4 activation.
Conversely, Pg-LPS failed to affect such locomotion.Ec-LPS treatment leads to elevated levels of IL-6, TNF-alpha and IL-10 in blood, likely mediated by TLR4 activation (Il-6 and IL-10) or other pathways distinct from activation of TAK-242sensitive TLR4 (TNF-alpha).Furthermore, Pg-LPS inhibits TLR4-mediated increases in IL-10 levels, while increased levels of IL-6 remained unaffected.The present study provides in vivo evidence that Pg-LPS can inhibit TLR4-mediated increases in IL-10, which is believed to prevent the development of periodontal disease.

Fig 1 .
Fig 1. Upper panel: Effects of intra-peritoneal injection of Pg-LPS on novel

Fig 3 .
Fig 3. Effects of intra-peritoneal injection of Ec-LPS on novel environment-

Fig 4 .
Fig 4. Effects of intra-peritoneal injection of Pg-or Ec-LPS with TAK-242 and

Fig 5 .
Fig 5. Effects of intra-peritoneal injection of Pg-or Ec-LPS with TAK-242 and

Fig 6 .
Fig 6.Effects of intra-peritoneal injection of Pg-or Ec-LPS with TAK-242 and