Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

We report here that the leptomeningeal cells transduce inflammatory signals from peripheral macrophages to brain-resident microglia in response to Porphyromonas gingivalis (P.g.) LPS. The expression of Toll-like receptor 2 (TLR2), TLR4, TNF-α, and inducible NO synthase was mainly detected in the gingival macrophages of chronic periodontitis patients. In in vitro studies, P.g. LPS induced the secretion of TNF-α and IL-1β from THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. Surprisingly, the mean mRNA levels of TNF-α and IL-1β in leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated RAW264.7 macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean mRNA levels of TNF-α and IL-1β in microglia after treatment with the conditioned medium from P.g. LPS-stimulated leptomeningeal cells were significantly higher than those after P.g. LPS alone. These observations suggest that leptomeninges serve as an important route for transducing inflammatory signals from macrophages to microglia by secretion of proinflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1 β production by leptomeningeal cells through inhibiting the nuclear factor-κB signaling pathway. Together with the inhibitory effect on microglial activation, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis.


Introduction
Periodontitis is the most common adult chronic inflammatory disorder, which results in a consequence of the persistent systemic inflammatory responses [1,2]. Porphyromonas gingivalis (P.g.) is the major periodontopathic bacteria [3,4], and its LPS (P.g. LPS) is thought to induce periodontitis through Toll-like receptors, TLR2 or TLR4 [5]. As the main population in inflammatory oral mucosa, macrophages are known to determine P.g. LPS-induced oral innate immune responses through TLRs during chronic periodontitis [5,6]. Macrophages can be polarized into M1 and M2 phenotypes depending on the microenvironment [7]. M1 macrophages promote inflammation and tissue damage by secreting proinflammatory mediators, including TNF-, IL-1 , and expressing inducible NO synthase (iNOS). In contrast, M2 macrophages promote anti-inflammation and wound healing by secreting anti-inflammatory mediators, including IL-10 and TGF-1, and upregulating arginase 1 (Arg 1) [8,9]. In addition to causing chronic systemic inflammatory diseases, including atherosclerosis, cardiovascular disease, and diabetes [10][11][12], periodontitis has been proposed as a risk factor for the central nervous system (CNS) disorders, such as Alzheimer's disease (AD) [13][14][15]. However, the exact route by which periodontitis transduces the peripheral inflammatory messages into the CNS remains unclear.
Besides the physical role as the cerebrospinal fluid-blood barrier [16,17], the leptomeninges also play roles as secretory cells, which transduce systemic inflammatory signals into the CNS [18][19][20]. Furthermore, TLR2 and TLR4 are detected in cultured human leptomeningeal cell lines [21] and leptomeninges of experimental animals [22,23], suggesting that leptomeninges are involved in the innate response of the CNS. Moreover, increasing evidence shows that microglia are the primary brain cells that respond to systemic inflammatory stimuli to play their well-known roles in neuroinflammation [24][25][26][27].
Propolis is a resinous substance produced by honeybees as a defense against intruders. It has relevant therapeutic properties that have been used since ancient times. The chemical composition of propolis depends on the local flora at the site of collection [28,29]. Considering its antioxidative and anti-inflammatory effects [30][31][32], propolis may have protective effects against neuroinflammatory responses.
In the present study, we have attempted to examine possible roles of leptomeninges in transducing inflammatory signals from peripheral macrophages to brain-resident microglia in response to P.g. LPS stimulation. The mean amounts of TNF-and IL-1 secreted by leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean amounts of TNF-and IL-1 secreted by microglia after treatment with the conditioned medium from P.g. LPStreated leptomeningeal cells were significantly higher than those after treatment with P.g. LPS alone. These observations suggest that leptomeninges transduce inflammatory signals from peripheral macrophages to brain-resident microglia by secreting inflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPSinduced TNF-and IL-1 production by leptomeningeal cells through inhibiting the nuclear factor-B (NF-B) signaling pathway. Together with our recent findings of direct inhibitory effects on the microglial inflammatory responses, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis.

Tissue Preparation from Periodontitis
Patients. The gingival samples were obtained from patients undergoing periodontal surgery or extraction. The periodontal diagnosis of subjects with chronic periodontitis was established based on clinical and radiographic criteria defined by the 1999 International World Workshop for a Classification of Periodontal Diseases and Conditions [33]. The samples included 9 cases diagnosed as chronic periodontitis (aged 34-60, 6 males and 3 females), which were recruited from Periodontology Department of School of Stomatology, Jilin University. Following surgery, excised gingival specimens were immediately placed in liquid nitrogen and subsequently frozen at −80 ∘ C until the following experiments.

Double-Immunofluorescent
Staining. The sections were hydrated and treated with 10% donkey serum for 1 h at 24 ∘ C and then were incubated with each primary antibody overnight at 4 ∘ C. The primary mouse monoclonal anti-TLR2 (T2.5, 1 : 200), mouse monoclonal anti-TLR4 (HTA-125, 1 : 200), goat polyclonal anti-TNF-(1 : 200), and mouse monoclonal anti-iNOS (4E5, 1 : 500) antibodies were mixed with rabbit polyclonal anti-Iba1 (1 : 500) antibody. After washing with PBS, the sections were incubated with a mixture of FITC-conjugated and rhodamine-conjugated secondary antibodies for 2 h at 24 ∘ C. After washing, the sections were mounted in the antifading medium Vectashield (Vector Laboratory) and examined by a confocal laser scanning microscope (CLSM) LSM510MET (CLSM, C2si, Nikon, Japan). CLSM images of individual sections were taken as a stack at 1 m step size along -direction with 20 × objectives (Numerical Aperture = 0.5), zoom factor 1.0. A rectangle (1024 × 1024 pixels) corresponding to the size of 450 × 450 m was used as the counting frame. CLSM images were shown as the middle of the stacked images.

Leptomeningeal Cell
Culture. Leptomeningeal cells were prepared from the brain of 3-day-old C57black/6N mouse.

Real-Time Quantitative RT-PCR Analysis.
THP-1 and RAW264.7 cells were treated with P.g. LPS (1 g/mL) for 24 h, leptomeningeal cells were incubated with the conditioned medium from P.g. LPS-or P.g. LPS-treated RAW264.7 cells (MCM) for 4 h, and MG6 were incubated with the conditioned medium from P.g. LPS-or P.g. LPS-treated leptomeningeal cells (LCM) for 24 h. The mRNA isolated from P.g. LPS-treated or nontreated cells were subjected to realtime quantitative RT-PCR. The total RNA was extracted with the Purelink RNA microkit (Invitrogen, Japan) according to the manufacturer's instructions. A total of 800 ng of extracted RNA was reverse transcribed to cDNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Foster City, CA). The thermal cycling was held at 50 ∘ C for 2 min, and then at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s and 60 ∘ C for 1 min. The cDNA was amplified in duplicate using TaqMan  For data normalization, an endogenous control (actin) was assessed to control for the cDNA input, and the relative units were calculated by a comparative Ct method. All real-time RT-PCR experiments were repeated three times, and the results are presented as the means of the ratios ± SEM.

ELISA Assay.
THP-1 and RAW264.7 cells were treated with P.g. LPS (1 g/mL), leptomeningeal cells were treated with P.g. LPS (100 ng/mL), and the condition medium was collected at 6 h, 24 h, 48 h, and 72 h after P.g. LPS treatment. RAW264.7 were incubated with propolis (15 g/mL) 1 h before P.g. LPS treatment, and the condition medium was collected at 48 h after treatment. The leptomeningeal cells were incubated with propolis (10 g/mL) 1 h before P.g. LPS treatment, and the condition medium was collected at 6 h after treatment. In the separated experiments, RAW264.7, leptomeningeal cells, and MG6 were treated with TLR2 (10 g/mL), TLR4 (10 g/mL) antibodies or the control antibodies or Bay 11-7082 (20 M) 1 h before P.g. LPS treatment. The condition medium was collected at the time points after the reagents treatment. TNF-and IL-1 released from THP-1, RAW264.7, leptomeningeal cells, and MG6 were measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) following the protocol provided by the manufacturer. The absorbency at 450 nm was measured using a microplate reader.
2.9. Determination of Cell Viability. RAW264.7 and leptomeningeal cells were seeded in 96-well plates for 24 h (5 × 10 3 cells/well) then incubated with various concentrations of propolis for 48 h. Cell viability was assessed using the Cell-Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, after propolis treatment, 10 L CCK-8 was added to each well and incubated at 37 ∘ C for 2 h. The optical density was read at a wavelength of 450 nm with a microplate reader. Cell viability was calculated using the following formula: optical density of treated group/control group × 100%.

Electrophoresis and
Immunoblotting. RAW264.7 and leptomeningeal cells were cultured at a density of 5 × 10 5 cells/mL, and the cytosolic samples of RAW264.7 and leptomeningeal cells were collected at 30 min, 60 min, and 120 min after P.g. LPS (1 g/mL, 100 ng/mL) treatment with or without propolis (15 g/mL, 10 g/mL). The samples were electrophoresed in 12% SDS-polyacrylamide gels, and the proteins on SDS gels were transferred electrophoretically to nitrocellulose membranes. Following the blocking, the membranes were incubated at 4 ∘ C overnight under gentle agitation with each primary antibody: rabbit anti-I B (1 : 1000), mouse anti-pI B (1 : 1000) antibodies. After washing, the membranes were incubated with horseradish peroxidase (HRP-) labeled anti-rabbit (1 : 2000, GE Healthcare, UK) or anti-mouse (1 : 2000, GE Healthcare, UK) antibodies for 2 h at 24 ∘ C, then the protein bands were detected by an enhanced chemiluminescence detection system (ECK kit, Amersham Pharmacia Biotech) using an image analyzer (LAS-4000, Fuji Photo Film, Tokyo, Japan).

Data
Analysis. The data are represented as the means ± SEM. The statistical analyses were performed using a oneway or two-way analysis of variance (ANOVA) with a post hoc Tukey's test using the GraphPad Prism software package.
A value of < 0.05 was considered to indicate statistical significance (GraphPad Software Inc., San Diego, CA, USA).

Characterization of Macrophages in Human Gingival
Tissues of Periodontitis and Cultured Macrophages after P.g. LPS Stimulation. We first examined the localization of TLR2, TLR4, and cytokines in human gingival tissues of chronic periodontitis patients, because macrophages are the main population in gingival tissues of chronic periodontitis to response P.g. LPS through TLR2 and TLR4 [6,38]. Our immunofluorescent double staining revealed that the immunoreactivities for TLR2, TLR4, TNF-, and iNOS corresponded well with those for Iba1 (Figure 1(a)) and their correspondence ratios were 72%, 79%, 53%, and 65%, respectively. However, immunoreactivities for IL-10 and TGF-1 were rarely found in human periodontitis gingival tissues (data not shown). We further determined the macrophage phenotypes after treatment with P.g. LPS using THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. In comparison to the nontreated cells, both the mean mRNA expression levels of TNF-and iNOS were significantly increased in THP-1 cells after treatment with P.g. LPS (1 g/mL). However, mean mRNA expression levels of IL-10 and Arg 1 were not significantly increased after treatment with P.g. LPS (Figure 1(b)). The similar results were also obtained in RAW264.7 cells (data not shown). Furthermore, the time-dependent release of TNF-and IL-1 from RAW264.7 cells was induced from 6 h and peaked at 48 h and then was decreased gradually later after P.g. LPS treatment (Figure 1(c)). The similar results were also obtained in THP-1 cells (data not shown). Moreover, P.g. LPS-induced TNF-and IL-1 production in RAW264.7 cells was significantly suppressed by anti-TLR2 antibody, but not by anti-TLR4 antibody (Figure 1(d)). On the other hand, the control antibodies with the same concentration had no significant effect (data not shown). These observations confirm that macrophages are polarized to M1 phenotype in response to P.g. LPS stimulation through TLR2.

Secretion of Proinflammatory Mediators by Leptomeni-
ngeal Cells after Treatment with the Conditioned Medium from P.g. LPS-Treated Macrophages and P.g. LPS. We next used mouse primary cultured leptomeningeal cells to address whether they could respond to inflammatory mediators secreted from P.g. LPS-treated macrophages using MCM and P.g. LPS alone. Surprisingly, the mean expression levels of TNF-and IL-1 mRNA in leptomeningeal cells were significantly increased from 4 h after treatment with MCM in comparison to those observed after treatment with P.g. LPS alone (Figure 2(a)). Furthermore, in comparison to the nontreated cells, the secretion of TNF-and IL-1 from leptomeningeal cells peaked at 6 h, decreased quickly, and it is noted that TNF-was undetected at 48 h after treatment with P.g. LPS (Figure 2(b)). Moreover, secretion of TNF-from leptomeningeal cells after treatment with P.g. LPS was significantly suppressed by anti-TLR2 antibody, but not by anti-TLR4 antibody (Figure 2(c)). On the other hand, the control antibodies with the same concentration had no significant effect (data not shown). To date, these observations provide the first evidence that leptomeningeal cells are polarized to proinflammatory phenotype in response to inflammatory signals from P.g. LPS-induced macrophages through TLR2.

Secretion of Proinflammatory Mediators by Microglia after Treatment with the Conditioned Medium from P.g. LPS-Treated Leptomeningeal Cells and P.g. LPS.
We have previously demonstrated that the leptomeninges are involved in the cytokine production by glial cells during chronic systemic inflammation [18][19][20]. In order to confirm that the leptomeninges could trigger microglial inflammatory responses, we next examined the mRNA expression of TNF-and IL-1 in MG6 microglia after treatment with the conditioned medium from P.g. LPS-treated leptomeningeal cells (LCM). The mean mRNA expression levels of TNF-and IL-1 were significantly increased after 24 h treatment with LCM in comparison to those observed after treatment with P.g. LPS alone (Figure 3(a)). We further examined the microglial responses after treatment with P.g. LPS, because P.g. LPS has been recently found in AD brain [15]. The mean level of TNF-secreted by microglia was significantly increased from 6 h and peaked at 48 h after treatment with P.g. LPS (100 ng/mL) in comparison to that by nontreated microglia. The amount of IL-1 secreted from microglia also reached peak at 48 h after treatment with P.g. LPS (data not shown). The P.g. LPS-induced secretion of TNF-by microglia was significantly suppressed by anti-TLR2 antibody, but not by anti-TLR4 antibody (Figure 3(b)). However, the control antibodies with the same concentration had no significant effect (data not shown). These observations clearly demonstrate that microglia were polarized to proinflammatory M1-like phenotype in response to inflammatory signals from P.g. LPSinduced leptomeningeal cells through TLR2.

Effect of Propolis on P.g. LPS-Induced Proinflammatory Phenotypes of Macrophages and Leptomeningeal Cells.
Finally, the effects of propolis on the secretion of P.g. LPS-induced proinflammatory mediators by macrophages and leptomeningeal cells were examined, because propolis has antioxidative and anti-inflammatory effects [31,32]. The mean cell viability was not significantly changed with the final concentrations until 15 g/mL on RAW264.7 macrophages (Figure 4(a)) and the final concentrations until 10 g/mL on leptomeningeal cells after treatment with propolis (Figure 4(d)). Therefore, we used propolis with the concentration of 15 g/mL on RAW264.7 and 10 g/mL on leptomeningeal cells, respectively, for the following experiments. In comparison to the nontreated cells, pretreatment with propolis significantly inhibited TNF-secretion by macrophages (Figure 4 with propolis significantly inhibited the P.g. LPS-induced phosphorylation of I B in RAW264.7 cells (Figure 4(c)) and leptomeningeal cells (Figure 4(f)). These observations demonstrate that propolis suppresses the P.g. LPS-induced proinflammatory responses by inhibiting the NF-B signaling pathway in both peripheral macrophages and leptomeningeal cells.

Discussion
The major findings of the present study are that leptomeninges transduce P.g. LPS-induced inflammatory signals from peripheral macrophages to brain-resident microglia, resulting in the induction of neuroinflammation. Furthermore, propolis was found to attenuate the secretion of P.g. LPS-induced proinflammatory mediators by leptomeningeal cells. To date, this is the first report to highlight that the leptomeninges serve as an important route for transducing peripheral inflammatory signals to the CNS during chronic periodontitis.
As the main population in inflammatory oral mucosa, macrophages phenotypes are known to determine P.g. LPSinduced oral innate immune responses through TLRs during periodontitis [5]. In the present study, we confirmed that macrophages densely expressed TLR2, TLR4, TNF-, and iNOS in the gingival tissues of chronic periodontitis patients. However, proinflammatory M1 macrophages are not limited to the infected gingiva but also increased in the circulation during chronic periodontitis [39,40]. The present observations indicate that P.g. LPS stimulation significantly increased the mean levels of mRNA expression of TNF-and iNOS, but not those of IL-10 and Arg1, suggesting that macrophages are polarized to M1 phenotype in response to P.g. LPS during chronic periodontitis. Furthermore, the mean amounts of P.g. LPS-induced TNF-and IL-1 secreted by macrophages were significantly suppressed by anti-TLR2 antibody, but not by anti-TLR4 antibody, further indicating that macrophages respond to P.g. LPS mainly through TLR2 [38,41,42], but not through TLR4 [5,42].

Mediators of Inflammation
Systemic inflammation and infections could worsen a number of CNS disorders [26,43]. Among the common chronic inflammatory disorders in adults, much attention has been paid to the periodontitis as the pathogenesis of CNS disorders, including AD [13,15,44]. The increase in macrophage-derived TNF-and IL-1 in the circulation during periodontitis [39,40] also supports the idea that chronic periodontitis is involved in the pathogenesis of systemic inflammatory diseases, including atherosclerosis, cardiovascular disease, and diabetes [10][11][12]. Therefore, it is reasonable to consider that the increased M1 polarization of macrophages during chronic periodontitis can be also a risk factor for AD, because the elevated levels of TNF-and IL1-are associated not only with the cognitive decline but also with the progression of AD [45][46][47]. Recently, we have reported that leptomeninges provide a critical link between chronic systemic inflammation and subsequent neuroinflammation [18][19][20]. In the present study, we have found that P.g. LPS stimulates both THP-1 and RAW264.7 macrophages to secrete TNF-and IL-1 . Furthermore, the mean mRNA expression levels of TNF-and IL-1 in leptomenigeal cells were significantly increased as early as 4 h after treatment with MCM. These observations indicate that leptomenigeal cells could respond to proinflammatory mediators secreted from M1 macrophages during chronic periodontitis. Furthermore, P.g. LPS also stimulates leptomeningeal cells to produce TNF-and IL-1 , which is consistent with previous studies using E coli LPS [18,19] and other meningitis causable agents [48][49][50]. Importantly, unlike peripheral macrophages, leptomeningeal cells produce TNF-and IL-1 from 6 h after treatment with P.g. LPS. Considering the inhibitory effect of TLR2 antibody on P.g. LPS-induced production of proinflammatory mediators, leptomeningeal cells could respond to P.g. LPS more sensitively than peripheral macrophages through TLR2, but not through TLR4, even though TLR4 is also expressed in meningeal cells [21,23].
Microglia are well-known key players of neuroinflammation [26,27], which are activated by TNF-and IL-1 in an autocrine manner [51,52]. The present findings indicate that LCM significantly enhanced the mRNA expression of TNFand IL-1 in microglia, suggesting that proinflammatory mediators secreted from P.g. LPS-treated leptomenigeal cells could subsequently activate microglia to generate neuroiflammation during chronic periodontitis. Furthermore, our present observations demonstrate that the mean levels of TNF-and IL-1 secreted by microglia were significantly increased after treatment with P.g. LPS alone. Furthermore, P.g. LPS-induced secretion of proinflammatory mediators by microglia was significantly suppressed by anti-TLR2 antibody. These observations support the recent idea that P.g. LPS may be involved in the progression of AD [15]. Furthermore, TLR2 is increased in peripheral blood mononuclear cells from AD patients [53]. Therefore, it is reasonable to consider that leptomenigeal cells may transduce the periodontitisderived inflammatory signals to microglia, resulting in powerful neuroninflammatory responses. Further investigations will be necessary to examine a possible involvement of other glial cells during chronic periodontitis, because other glial cells, such as astrocytes, also contribute to the CNS disorders [54,55]. Moreover, further investigations are necessary to clarify the involvement of other factors in neuroinflammation during chronic periodontitis, because P.g. LPS is only one of the related factors of chronic periodontitis. Neuroprotective drug therapies have not yet translated well from the lab to the clinic because of an excessive focus of treatments on promoting the survival of neurons, with far less work on nonneuronal brain cells. Recently, leptomeninges have been focused on delivering compounds/genes to brain [56,57]. Therefore, meninges can be considered as the direct targets for treating CNS disorders, including AD. Propolis is a resinous substance produced by honeybees as a defense against intruders. It has relevant therapeutic properties that have been used since ancient times. Depending on their antioxidative and anti-inflammatory effects [30][31][32], we here provide the first evidence that propolis can significantly inhibit TNF-and IL-1 production by both RAW264.7 cells and leptomenigeal cells through inhibiting the NF-B signaling pathway, because NF-B is a critical transcription factor that encodes genes of proinflammatory mediators, including TNF-and IL-1 [58]. These findings agreed well with our previous observations that propolis significantly inhibited hypoxia-induced NF-B-dependent production of proinflammatory mediators by microglia. Recently, we have reported that the exaggerated neuroinflammatory responses evoked by microglia are responsible for an impairment of the hippocampal long-term potentiation in the middle-aged animals subjected to adjuvant arthritis [59]. Therefore, the efficient attenuation of propolis in P.g. LPS-induced NF-Bdependent proinflammatory pathway of leptomeningeal cells and microglia may prevent the age-dependent exaggerated neuroinflammatory responses in the CNS.

Conclusion
In conclusion, our present findings strongly suggest that the leptomeninges serve as an important route for transducing inflammatory signals from peripheral macrophages into brain-resident microglia by secreting proinflammatory mediators during chronic periodontitis. Propolis may benefit for preventing and reducing neuroinflammation in CNS disorders, including AD, by attenuating P.g. LPS-induced inflammatory signals from peripheral macrophages, leptomeningeal cells and microglia during chronic periodontitis. Further investigations are necessary to clarify the involvement of other factors in neuroinflammation during chronic periodontitis.