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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2014 Aug;96(2):349–357. doi: 10.1189/jlb.4A0214-095R

B cells promote obesity-associated periodontitis and oral pathogen-associated inflammation

Min Zhu *, Anna C Belkina *, Jason DeFuria *, Jordan D Carr *, Thomas E Van Dyke †, Robert Gyurko ‡, Barbara S Nikolajczyk *,1
PMCID: PMC4101090  PMID: 24782490

B cells promote bone loss and inflammation in obesity-associated periodontitis, but play a modest role in periodontitis of lean hosts.

Keywords: B lymphocyte, type 2 diabetes, mouse model, Porphyromonas gingivalis, cytokine

Abstract

Individuals with T2D and PD suffer significantly from the ability of one disease to intensify the other. Disease-associated inflammation is one mechanism thought to fuel this pathogenic feed-forward loop. Several lines of evidence indicate that proinflammatory B cells promote T2D and PD; thus, B cells are top candidates for a cell type that predisposes PD in T2D. To test directly the role of B cells in T2D-associated PD, we compared outcomes from oral Porphyromonas gingivalis challenge of lean WT or B cell-null mice with outcomes from mice that were obese and insulin-resistant before challenge. Obese WT mice responded to oral P. gingivalis challenge with significant periodontal bone loss, whereas obese B cell-null mice were protected completely from PD. By contrast, lean WT and B cell-null mice suffer similar periodontal bone loss in response to oral pathogen. B cells from obese/insulin-resistant hosts also support oral osteoclastogenesis and both oral and systemic production of inflammatory cytokines, including pro-osteoclastogenic TNF-α and MIP-2, an ortholog of human IL-8. B cells furthermore impact AT inflammation in obese, P. gingivalis-infected hosts. Taken together, these data show that fundamentally different mechanisms regulate PD in lean and obese hosts, with B cells able to promote PD only if the hosts are “primed” by obesity. These results justify more intense analysis of obesity-associated changes in B cells that predispose PD in human T2D.

Introduction

PD is a chronic inflammatory disease, characterized by destruction of tooth-supporting soft and hard tissue (periodontium and bone), and is among the most prevalent health conditions in the world [1]. PD is strongly linked to systemic changes, as evidenced by the list of associated diseases that includes T2D [2–4]. Patients who develop T2D and PD suffer significant morbidity from the seemingly cyclical nature of one disease confounding the other [3], but mechanisms underlying the relationship between these diseases remain poorly defined. One unifying link between T2D and PD is unresolved inflammation. Therefore, the field widely supports the identification of common inflammatory mediators in T2D and PD as a strategy that will pave the way for development of novel therapeutics aimed to alleviate both chronic diseases simultaneously.

B cells have long been linked to PD in lean humans and in animal models. B cells infiltrate and dominate the site of oral infection following an initial immune response by neutrophils and monocytes [5], and the proportion of B cells in oral lesions positively correlates with disease severity in PD patients [6]. Adoptive transfer studies indicated more definitively that B cells play a role in PD, as transfer of oral pathogen-primed B cells promotes periodontal bone loss in the recipient animal once the recipient is exposed to the same pathogen [7]. However, the artificial priming approach in these PD studies does not address whether in situ B cells play similarly important roles. Although early studies suggested that B cells protect the host from PD through the unique ability to produce antibodies specific for PD pathogens, the role of the pathogen-specific antibodies in disease remains highly controversial, given that antibodies either positively or negatively correlate with clinical disease [8–10]. More recent work has shown that human B cells are major sources of cytokines implicated in periodontal bone loss, including TNF-α, IL-8, and IL-1β [11]. Furthermore, up to 90% of B cells in periodontal lesions express RANKL, a factor required for osteoclast differentiation/survival, thus, periodontal bone resorption [12]. Overall, regardless of myriad mechanisms that may link B cells with PD, no studies have definitively identified the overall in situ roles of B cells in PD pathogenesis.

Data from multiple labs also indicate that B cells play important roles in T2D. Studies of human samples showed that insulin-resistant (presumably prediabetic or T2D) individuals have increased titers of serum autoantibodies [13]. Subsequent murine studies demonstrated that IgG from diet-induced obese mice but not from lean mice induced abnormal glucose metabolism in recipient B cell-null mice, consistent with the possibility that obesity triggers production of pathogenic Igs [13]. However, autoreactivity of IgGs from obese mice remains controversial [14]. B cells from T2D patients also secrete relatively high concentrations of proinflammatory IL-8 and perhaps more importantly, are unable to produce significant amounts of anti-inflammatory IL-10 [15]. In vivo studies showed that obese B cell-null mice have decreased systemic and AT inflammation and are more insulin-sensitive than obese WT mice [13, 14]. Thus, B cells function to promote obesity-associated metabolic disease. Overall, marked similarities between disease-associated changes in B cell function in T2D and PD outlined above raise the possibility that B cells are important components of the inflammatory process that mediate increased incidence of PD in T2D.

To identify the role of B cells in PD pathogenesis, we induced PD using a standard P. gingivalis oral inoculation method in lean WT or B cell-null mice. Surprisingly, we found that B cells play an insignificant role in PD in lean mice. However, serial induction of obesity and PD in WT and B cell-null mice demonstrated that obesity-associated changes in B cell function increased B cell response to oral pathogen ligands and potentiated pathogen-induced oral bone loss. These data indicate that fundamentally different mechanisms control PD in lean and obese mice: PD development is B cell-independent in leans, yet requires B cells in mice with pre-existing obesity and insulin resistance. Taken together, these data raise the radical possibility that B cell depletion may effectively break the feed-forward inflammatory loop between T2D and PD.

MATERIALS AND METHODS

Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee of Boston University and performed in conformance with the standards of the Public Health Service Policy on Humane Care and Use of Laboratory Animals. WT or B cell-null (μMT) [16] mice on a C57BL/6J background were ordered from The Jackson Laboratory (Bar Harbor, ME, USA) or were bred in-house, and all were maintained on-site in sterile cages. Results from B cell-null mice (verified by absence of CD19+ B cells in flow cytometry), bred in-house or at The Jackson Laboratory, were indistinguishable.

Establishment of a T2D-associated PD model

Five-week-old male mice were fed ad libitum with a LFD (10% energy from fat; Research Diets #D12450Bi; micronutrient matched to the HFD) or a HFD (60% energy from fat; Research Diets #D12492i) for 10 weeks before oral inoculation detailed below (see Fig. 1A). Weight-matched WT and B cell-null mice were assigned to P. gingivalis- or sham-infected groups and received standard oral inoculation following antibiotic treatment as outlined below.

Figure 1. Oral inoculation ameliorates the genotype-associated differences of systemic metabolic variables in obese mice.

Figure 1.

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(A) Schematic representation for the combined diet-induced obesity and P. gingivalis (P.g) oral inoculation model. WT or B cell-null mice were fed a HFD or LFD, starting at 5 weeks of age for a total of 16 weeks. Inoculated groups were treated with oral antibiotics at 7.5 weeks of the diet and carboxymethylcellulose (vehicle) or P. gingivalis (P.g) at 10 weeks of the diet. (B) Weekly body-weight gain of HFD- and LFD-fed mice. Data with different oral inoculation treatment are combined and presented as mean ± se; P < 0.05, n = 16–18/group. Error bars are obscured by symbols at most points. (C) Six-hour-fasting blood glucose of oral-inoculated mice after 14–15 weeks on a HFD. Data are presented as mean ± se; n = 6–9/group. (D) Six-hour-fasting blood glucose of mice after 14–15 weeks on a HFD without antibiotics or oral inoculation treatment. Data are presented as mean ± se; ***P < 0.001, n = 6–9/group. (E) GTT of oral-inoculated mice after 14–15 weeks on a HFD. Differences among conditions are insignificant (P>0.05), but results are split into two panels for clarity. Data are presented as mean ± se, n = 6–9/group. (F) GTT of mice after 14–15 weeks on a HFD without antibiotics or oral inoculation treatment. Data are presented as mean ± se, n = 6–9/group. **P < 0.01, and ***P < 0.001, comparison between blood glucose of WT and B cell-null mice at the same time-point. Area under the curve analysis confirmed the difference between noninoculated WT and B cell-null mice (P < 0.05).

Frozen stocks of P. gingivalis strain A7436 were grown anaerobically at 37°C on CDC blood agar plates (BD Biosciences, San Jose, CA, USA) for 3–5 days. Plate-grown organisms were used to inoculate liquid cultures of Schaedler broth (BD Biosciences) and were grown anaerobically for 18–24 h and then harvested at mid- to late-log phase.

LFD- and HFD-fed mice were treated with the broad-spectrum antibiotic combination of sulfamethoxazole (0.8 mg/ml) plus trimethoprim (0.16 mg/ml; Sulfatrim; HiTech Pharmacal, Amityville, NY, USA) ad libitum in their drinking water for 10 days after 7.5 weeks of diet, followed by a 3-day antibiotic-free period. After 10 weeks on the diet (15 weeks of age), mice were infected by oral, topical application of P. gingivalis at the buccal and palatal surface of the maxillae, three times on alternate days (Monday, Wednesday, and Friday) with 109 CFU live P. gingivalis in 100 μl vehicle consisting of PBS plus 2% carboxymethylcellulose as a carrier [17] or vehicle alone. Six weeks later (16 weeks on the diet; 21 weeks of age), mice were killed by CO2 narcosis/cervical dislocation. Alternatively, 5- to 6-week-old chow-fed, lean females were treated with antibiotics and then oral inoculation to induce PD, as published previously [17]. Serum was collected in BD Microtainer tubes (BD Biosciences). Tissues for sectioning were fixed in Z-Fix (Anatech, Battle Creek, MI, USA) postharvest and then stored in PBS. Tissues for mRNA assays were snap-frozen in liquid nitrogen immediately postharvest and stored at −80°C.

Metabolic measures

Body weights were recorded periodically for LFD- and HFD-fed mice. GTTs were performed on mice at 8–9 and 14–15 weeks on the diet. For GTT, mice were fasted for 6 h and then injected i.p. with 1 g/kg sterile D-glucose. Blood glucose was monitored from tail vein blood at the indicated times using an automated glucometer.

Periodontal bone-loss measures

Freshly dissected maxilla was defleshed in a dermestid beetle colony and stained with methylene blue (1% in water) prior to photographing. Digitalized images of the maxillary molars were analyzed with MicroSuite (Olympus America, Melville, NY, USA). The CEJ-ABC distance was measured by a blinded analyst to indicate periodontal bone loss, a key feature of PD. Mean values of 14 sites that included all six upper molars were reported for each animal. The total area of alveolar bone loss was also quantified. Differences between P. gingivalis-inoculated and control mice were interpreted as periodontal bone loss.

Histomorphometry and IHC

Fixed mandibles were demineralized in 10% EDTA in 0.1 M Tris buffer and embedded in paraffin before thin sectioning (6μ M) in a mesio-distal direction. Sections were stained with TRAP for 90 min, followed by hematoxylin counterstain [18]. Digital images were acquired by the Olympus Upright microscope, and osteoclasts were counted in deidentified sections as multinucleated, dark red cells along the alveolar crest. Sections were also stained with anti-mouse B220 (1:1000; R&D Systems, Minneapolis, MN, USA) or rat IgG (control antibody, 1:1000) and counterstained with hematoxylin.

Fixed epididymal fat pads were embedded in paraffin and step-sectioned. Sections (5 μM) were stained with the following antibodies: anti-mouse F4/80 (1:10,000; Cedarlane Laboratories, Burlington, NC, USA), anti-mouse B220 (1:1000; R&D Systems), or rat IgG, and counterstained with hematoxylin. Digital images were acquired by an Olympus Upright microscope. F4/80-positive crown-like structures [19] were quantified as numbers of macrophage-rich, crown-like structures per number of adipocytes in the field.

Quantitative PCR and flow cytometry

RNA was extracted using a Mini-Beadbeater (Biospec, Bartlesville, OK, USA) and an RNeasy lipid tissue mini kit (Qiagen, Valencia, CA, USA) and quantified based on a A260:A280 ratio. Total RNA (1 μg) was reverse-transcribed using the RT2 First Strand Kit (Qiagen), and cDNA was amplified on custom-designed, 384-well RT2 Profiler PCR Array Plates (Qiagen) with proprietary primers. Target genes were normalized to the arithmetic mean of two housekeeping genes, Hprt1 and Rplp0, by the 2−ΔΔ comparative threshold method, and all data are reported as relative to the control group as specified. Foxp3 staining of splenocytes for regulatory T cell analysis by flow cytometry was performed as published previously [14].

Multiplex protein assay

Total splenocytes were isolated and incubated for 24 h in vitro in media alone or with Pam3Csk (1 mg/ml; InvivoGen, SanDiego, CA, USA), P. gingivalis LPS (1 mg/ml; InvivoGen), CpG (1 μg/ml; InvivoGen), or anti-BCR (1 mg/ml; Jackson ImmunoResearch, West Grove, PA, USA), with anti-CD40 (1 mg/ml; eBioscience, San Diego, CA, USA), as indicated. Cytokines were quantified in supernatants using multiplex cytokine analyses (Millipore, Billerica, MA, USA), a BioPlex 200 analyzer (BioRad, Hercules, CA, USA), and the BioPlex Manager 5 program.

Statistics

All data are reported as mean ± se unless indicated otherwise. Comparisons between two groups were analyzed using Student's two-tailed unpaired t-test. The effect of genotype and oral inoculation treatment was determined by two-way ANOVA with Tukey's “Honestly Significant Difference” test. Statistical significance was reported at P < 0.05.

RESULTS

B cells promote PD in obese/glucose-intolerant mice

B cells from T2D and PD patients secrete a similar proinflammatory cytokine profile [11, 15], which taken together with the possibility of pathogenic antibodies [13], suggests that B cells may play critical roles in T2D-associated PD. To develop a model to test this possibility, we induced obesity in WT and B cell-null mice by an ad libitum HFD feeding (Fig. 1A), which predictably resulted in weight gain (Fig. 1B and Supplemental Fig. 1A), high fasting blood glucose (Fig. 1C and Supplemental Fig. 1B), glucose intolerance, and insulin resistance (Fig. 1E and Supplemental Fig. 1C–E) compared with LFD-fed mice. In contrast to the mild protection from elevated blood glucose and glucose intolerance in obese B cell-null mice compared with obese WT mice, as we have published previously (Fig. 1D and F and Supplemental Fig. 1F) [14], WT and B cell-null mice, pretreated with antibiotics in preparation for P. gingivalis oral inoculation, had comparable fasting concentrations of blood glucose and were similarly glucose-intolerant at midway (8–9 weeks on the diet; Supplemental Fig. 1E) and late (14–15 weeks on the diet; Fig. 1C and E) time-points in the feeding protocol. These data demonstrate that any differences in periodontal pathogen response of obese WT and B cell-null mice are independent of measurable metabolic differences.

To test the role that B cells play in T2D-potentiated PD, we measured P. gingivalis-induced bone loss in obese mice from WT and B cell-null genotypes. Morphometric analyses of CEJ-ABC distance/area showed that oral P. gingivalis inoculation induced periodontal bone loss in obese WT mice (Fig. 2A and B). In sharp contrast to the response of obese WT mice to P. gingivalis, B cell-null mice are completely protected from P. gingivalis-induced bone destruction (Fig. 2A and B). These data support the conclusion that B cells promote PD in the context of obesity and glucose intolerance.

Figure 2. B cells promote P. gingivalis-induced periodontal bone loss in obese/glucose-intolerant mice.

Figure 2.

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(A) Vertical alveolar bone loss of HFD-fed mice after oral inoculation was determined by measuring the CEJ-ABC distance. Data are presented as mean ± se, n = 8–9/group; *P < 0.05. (B) Alveolar bone loss area of HFD-fed mice after oral inoculation was determined by measuring the total area between CEJ and ABC. Data are presented as mean ± se, n = 8–9; *P < 0.05.

B cells play insignificant roles in PD of lean hosts

Several studies suggest that B cells regulate PD outside of the context of obesity. To test this possibility definitively, we orally inoculated lean WT or B cell-null mice with P. gingivalis to induce PD and then measured periodontal bone loss. Surprisingly, P. gingivalis-induced periodontal bone loss was similar in lean WT and B cell-null mice, regardless of whether the mice were relatively young, lean females on normal chow (P>0.05 comparing the WT P. gingivalis group and the B cell-null group; Fig. 3A) or LFD-fed, “middle-aged” males (P>0.05 comparing the WT P. gingivalis group and the B cell-null group; Fig. 3B and C). These data support the interpretation that B cells play a minor, if any, role in PD of lean hosts. Comparison of outcomes from lean and obese mice (Figs. 2A vs. 3B and Figs. 2B vs. 3C) supports the unexpected conclusion that B cells play a fundamentally different role in PD and T2D-potentiated PD.

Figure 3. B cells play insignificant roles in PD of lean hosts.

Figure 3.

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(A) Five-week-old WT or B cell-null mice fed on normal chow were subjected to the P. gingivalis-induced PD protocol, and vertical alveolar bone loss was determined by measuring the CEJ-ABC distance. Data are presented as mean ± se, n = 8–9/group; *P < 0.05. (B) Vertical alveolar bone loss of LFD-fed mice after oral inoculation was determined by measuring the CEJ-ABC distance. Data are presented as mean ± se, n = 8–9/group. **P < 0.01, and ***P < 0.001. (C) Alveolar bone loss area of LFD-fed mice after oral inoculation was determined by measuring the area between CEJ and ABC. Data are presented as mean ± se, n = 8–9/group; **P < 0.01, and ***P < 0.001. Differences between WT P. gingivalis and B cell-null P. gingivalis were insignificant in all panels.

B cells promote local inflammation in obesity-associated PD

To measure the role that B cells play in the oral bone destruction characteristic of obesity-associated PD, we used TRAP staining to identify and quantify the presence of bone-resorptive osteoclasts adjacent to alveolar bone in obese mice. The number of alveolar bone-lining osteoclasts per mm bone was significantly higher in obese WT compared with obese B cell-null mice following P. gingivalis inoculation (Fig. 4A). Thus, the increased number of osteoclasts mechanistically explains the overall higher oral bone loss in obese WT compared with obese B cell-null mice.

Figure 4. B cells promote osteoclastogenesis and periodontal inflammation in obesity-associated PD.

Figure 4.

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(A, left) Representative images of TRAP staining of periodontal tissue of obese, P. gingivalis-inoculated mice (n=8/group); arrows show the TRAP-positive osteoclasts; scale bars represent 0.1 mm. (Right) Quantification of alveolar-lining osteoclasts as number of osteoclast/linear distance of alveolar surface; data are presented as mean ± se, n = 8/group; *P < 0.05. (B) mRNA expression of TNF-α in gingival tissue. Data are presented as mean ± se, n = 8–9/group. **P < 0.01. (C) mRNA expression of RANKL in gingival tissue. P = 0.1 when comparing WT vehicle and WT P. gingivalis group. Data are presented as mean ± se, n = 8–9/group. (D) mRNA expression of Foxp3 in gingival tissue. Data are presented as mean ± se, n = 8–9/group. **P < 0.01.

Multiple proinflammatory cytokines, including RANKL and TNF-α, contribute to the differentiation and activation of osteoclasts [20]. To test the possibility that gingiva from obese WT and obese B cell-null mice up-regulate pro-osteoclastogenic cytokines in response to oral pathogen challenge, we quantitated expression of pro-osteoclastogenic cytokines in gingival tissue of these mice by RT-PCR. Gingiva of P. gingivalis-infected, obese WT, but not obese B cell-null mice, had higher TNF-α expression (Fig. 4B) and a trend toward increased RANKL expression compared with gingiva from mock-infected, obese animals (Fig. 4C). Given that B cells secrete cytokines directly but may additionally/alternatively coordinate other immune cells for cytokine secretion, we used IHC and RT-PCR arrays to identify the cell types that may account for increased TNF-α and RANKL in gingiva of obese WT (but not B cell-null) mice responding to oral pathogen challenge. B220-specific IHC showed that B220+ B cells were present in the periodontal tissue (Supplemental Fig. 2A, left) but were rare in gingiva of obese WT mice (Supplemental Fig. 2A, right) at the study endpoint, 6 weeks post-P. gingivalis inoculation. RT-PCR arrays of gingival tissue showed extremely low CD19 mRNA expression (data not shown), which was consistent with IHC data. Furthermore, we saw no significant difference in mRNA expression of T cell markers (CD4, CD8), costimulatory markers (CD80, CD86), or the macrophage activation marker F4/80 in gingiva of obese, P. gingivalis-infected WT and B cell-null mice (data not shown). Interestingly, Foxp3 mRNA, a marker for anti-inflammatory regulatory T cells, was similarly expressed in gingiva of obese, P. gingivalis-infected WT and B cell-null mice, but its expression was decreased significantly in response to P. gingivalis infection in B cell-null mice (Fig. 4D). We conclude that B cells are required for gingival expression of osteoclastogenic cytokines in obese hosts, although B cells are only present in subgingival periodontal tissue 6 weeks post-PD induction.

B cells promote systemic inflammation in obesity-associated PD

Our previous work showed that B cells from PD or T2D subjects secrete a proinflammatory cytokine profile that may directly contribute to the chronic inflammation that characterizes both conditions [11, 15]. To test the possibility that B cells contribute to chronic systemic inflammation in T2D-associated PD, we measured the potential of splenic B cells from obese, P. gingivalis-inoculated mice to produce a proinflammatory/osteoclastogenic cytokine profile in response to TLR ligands, including TLR-activating P. gingivalis LPS [21]. Importantly for these studies, the vast majority (80–90%) of robust TLR responders in C57BL/6 splenocytes are B cells. Unstimulated splenocytes from obese mice produced little TNF-α, with concentrations that were largely under the level of detection as expected (data not shown). Splenocytes from P. gingivalis-inoculated, obese WT mice responded to the TLR2 ligand Pam3Csk4 by secreting increased concentrations of TNF-α compared with cells from control-inoculated WT mice or obese B cell-null mice (Fig. 5A). Unexpectedly, the TLR2 ligand from P. gingivalis LPS elicited little TNF-α response in all cultures, uncovering ligand-dependent differences in P. gingivalis-primed TLR2 responses (data not shown). To test further the role that B cells play in P. gingivalis-potentiated TNF-α production, we stimulated splenocytes with the B cell-activating TLR9 ligand CpG, which similarly elicited more TNF-α production by splenocytes from obese, P. gingivalis-inoculated WT versus splenocytes from control-inoculated WT or B cell-null mice (Fig. 5B). Finally, splenocyte stimulation with anti-BCR (immunoglobulin M.) plus anti-CD40 resulted in genotype and inoculation-independent TNF-α production (Fig. 5C). These data support the conclusion that in vivo periodontal pathogen exposure in obesity promotes high TNF-α production in B cells responding to a select subset of TLR agonists.

Figure 5. B cells support TNF-α production by splenocytes responding to TLR ligands after in vivo periodontal pathogen exposure.

Figure 5.

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Splenocytes from obese, oral-inoculated mice were stimulated with (A) Pam3Csk4, (B) CpG, or (C) anti-BCR and anti-CD40 for 24 h. Shown is TNF-α in culture supernatants. Data are presented as mean ± se, n = 8–9/group. *P < 0.05, and **P < 0.01.

Similar studies tested the possibility that in vivo P. gingivalis exposure primes B cells from obese mice to produce MIP-2, the murine ortholog of human IL-8, a chemokine that is strongly implicated in PD and T2D [11, 15, 22]. Unstimulated splenocytes from obese mice constitutively produced similar concentrations of MIP-2 after 24 h in culture, regardless of genotype or inoculation treatment (Fig. 6A). However, stimulation with the TLR2 ligands Pam3Csk4 and P. gingivalis LPS elicited two- to threefold more MIP-2 from splenocytes of obese WT mice exposed to P. gingivalis compared with cells from vehicle-inoculated mice (Fig. 6B and C). Importantly, the same number of splenocytes from B cell-null mice produced relatively low concentrations of MIP-2 in response to TLR2 ligands, irrespective of whether mice were in vivo-treated with P. gingivalis or carrier (Fig. 6B–D). In contrast, stimulation with a combination of anti-BCR plus anti-CD40 elicited background levels of MIP-2 from all splenocytes tested (Fig. 6E). These data are consistent with the interpretation that in vivo periodontal pathogen exposure in T2D primes B cells to support higher MIP-2 production in response to TLR restimulation.

Figure 6. B cells support MIP-2 production by splenocytes responding to TLR ligands after in vivo periodontal pathogen exposure.

Figure 6.

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(A) Splenocytes from obese, oral-inoculated mice were cultured in media alone for 24 h, and MIP-2 in supernatant was quantified. Alternatively, splenocytes from obese, oral-inoculated mice were stimulated with (B) Pam3Csk4, (C) P. gingivalis LPS, (D) CpG, or (E) anti-BCR and anti-CD40 for 24 h. Shown is MIP-2 in culture supernatants. Data are presented as mean ± se, n = 8–9/group. *P < 0.05, and ***P < 0.001.

B cells impact AT inflammation in obesity-associated PD

Given that AT is a major contributor to inflammation in obesity/T2D, and lack of B cells decreases AT inflammation in a standard obesity model [14], we tested the possibility that B cells also impact AT inflammation in obesity-associated PD. As macrophages are the dominant cell type in inflamed AT from obese mice, we first performed F4/80 staining for activated macrophages in eAT from the variously treated mice. Quantification of macrophages, as evidenced by macrophage-rich, crown-like structures (Fig. 7A and Supplemental Fig. 2B, arrows) [19], revealed a significant reduction in eAT macrophages from obese B cell-null mice inoculated with P. gingivalis- compared with mock-infected counterparts. However, no significant differences in macrophage-rich, crown-like structures were detected in eAT from obese WT mice regardless of inoculation treatment (Fig. 7A and Supplemental Fig. 2B). The results indicate that eAT in obese B cell-null mice is unexpectedly less inflamed in response to P. gingivalis.

Figure 7. B cells impact eAT inflammation in obesity-associated PD.

Figure 7.

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(A) Macrophage-rich crown-like structures in eAT of HFD-fed, oral-inoculated mice were identified by F4/80 immunostaining and quantified as number of crown-like structures/number of adipocytes in the field. Importantly, adipocyte size was similar among all mice (not shown). mRNA expression of (B) CD19, (C) Foxp3, or (D) IL-1β, IL-5, Retnla, and Klrb1c in eAT or spleen of obese, oral-inoculated mice, as indicated. Data are presented as mean ± se, n = 8–9/group. *P < 0.05, **P < 0.01, and ***P < 0.001.

We explored further signatures of AT inflammation on RT-PCR array analysis of eAT. Similar levels of CD19 mRNA expression were detected in eAT from obese WT mice regardless of inoculation treatment (Fig. 7B). This result is consistent with similar B220+ cell infiltration into the eAT, as demonstrated by IHC (Supplemental Fig. 2C), and the similar number of crown-like structures in these tissues (Fig. 7A). CD19 mRNA expression was undetectable in eAT from B cell-null mice (Fig. 7B), as expected. Importantly, Foxp3 mRNA (Fig. 7C, left) was expressed at significantly higher amounts in P. gingivalis-inoculated, obese B cell-null mice compared with the vehicle-inoculated controls. In contrast, Foxp3 was similarly expressed in eAT from P. gingivalis- and vehicle-inoculated, obese WT mice. Foxp3 mRNA (Fig. 7C, right) or Foxp3+ CD4+ cells (data not shown) were similar in the splenocytes of obese mice regardless of genotype and inoculation treatment, which is consistent with the finding that regulatory T cells in AT are distinct from those in peripheral lymphoid organs [23]. The notion that B cells promote an inflammatory phenotype was evidenced further by significantly decreased mRNA expression of proinflammatory IL-1β and increased expression of anti-inflammatory IL-5 and Retnla in eAT from obese, P. gingivalis-infected B cell-null mice compared with obese, P. gingivalis-infected WT mice (Fig. 7D). Interestingly, mRNA expression of NK cell marker Klrb1c was significantly lower in obese, P. gingivalis-infected B cell-null mice (Fig. 7D). We conclude that B cells influence the response of eAT to T2D-associated PD.

DISCUSSION

Findings herein show that B cells play critical roles in obesity-associated PD but play a modest, if any, role in PD of lean hosts. The roles of B cells in obesity-confounded PD are evidenced further by the increased osteoclasts and major osteoclastogenic cytokines, most convincingly, TNF-α, in obese, P. gingivalis-inoculated WT compared with B cell-null mice. In addition to gingival inflammation, B cells promote systemic inflammation in an obesity-associated PD model by supporting proinflammatory TNF-α and MIP-2 production following priming with oral P. gingivalis, although some tissues, such as eAT, may be exempt from P. gingivalis-associated inflammation in the absence of B cells. Overall, the data indicate that B cells promote inflammation in T2D-associated PD, at least in part, through supporting osteoclastogenic and proinflammatory cytokine secretion.

Recent studies support a potential three-way relationship among obesity, T2D, and PD, with each condition influencing the others [24]. T2D-associated changes, including the excess soluble advanced glycation products, are shown to be specific cell-activating molecules that aggravate the chronic inflammation of periodontal disease [25, 26]. However, the effector cells are less explored, despite the fact that cells could be concrete targets for breaking the feed-forward loop. One animal study showed that macrophages in obese mice respond to P. gingivalis with lower inflammatory cytokine production compared with macrophages from leans [27], which contradicts most of currently available work on immune cell function in T2D-associated PD showing an overall hyper-responsive state. By contrast, lymphocytes, especially the B cells that dominate PD lesions, are strongly implicated in obesity-associated PD [24] through newly appreciated pro-inflammatory functions that may be more amenable to clinical perturbation.

The first step toward testing the role B cells play in obesity-associated PD was to establish a model of PD induction in obese mice. Previous mouse models of obesity-associated PD have raised questions in the immunometabolism community, including how fasting glucose measurements can be similar in lean and obese mice [27]. Although our analyses agree with the broader literature, which shows that all obese WT mice become hyperglycemic in response to diet-induced obesity, our data unexpectedly indicate that the standard oral inoculation protocol eliminates genotype-associated differences in fasting blood glucose and glucose tolerance in the obese mice. Given that (1) the standard oral inoculation protocol includes treatment with a broad-spectrum antibiotic to optimize P. gingivalis colonization in mice, (2) the whole-body metabolic changes we reported are pathogen-independent, and (3) the microbiota play a critical role in metabolic health [28, 29], we speculate that the broad-spectrum antibiotic treatment impacts metabolic parameters by significantly shifting murine flora. Although a full microbiota analysis goes beyond the focus of the current work, this newly appreciated aspect of the model will be helpful for the field to assess T2D-associated PD in the absence of gross metabolic differences in, for example, immunologically compromised (especially knock-out) animals.

Given the rich literature linking B cells to PD [30, 31], our data unexpectedly support the conclusion that naturally occurring B cells play a minor, if any, role in the development of PD in lean hosts. With the unique capability of generating antibodies, B cells were thought to alleviate or exacerbate PD with anti-pathogen antibodies [9, 32, 33]. It was also possible that B cells regulate PD in leans through a proinflammatory cytokine profile or through their ability to regulate T cell function [34]. Nevertheless, our study in lean B cell-null and WT mice with PD suggest that these protective and/or destructive components of B cells are irrelevant or, perhaps more likely, cancel each other out to result in an insignificant impact of the B cell in PD pathogenesis outside of the obesity milieu. These findings must be tested for relevance to humans to determine whether further work on B cells in lean individuals is justified.

B cells secrete cytokines and coordinate other immune cells in tissue inflammation [35, 36], even though B cells may enter and exit the tissue (or die) at early stages in disease development. One example of this phenomenon is the transient peak of B cells in the expanding eAT during the development of obesity [13, 14, 37] and the subsequent paucity of eAT B cells later in disease. Transient influx of B cells in AT may have initially undermined work on the proinflammatory roles that B cells are now known to play in longer-term metabolic disease [4]. Although temporal analyses will be needed to determine whether B cells similarly infiltrate infected gingiva in a transient wave that peaks well before our 6-week post-inoculation end-point, findings herein are consistent with the possibility that B cells set the stage for obesity-associated PD but are less important later in chronic disease. The mouse model of T2D-associated PD may therefore be helpful for studies of PD etiology, while later phases of PD may be better studied in a model characterized by B cell-dominated lesions that mimic long-term human disease. Further studies are needed to identify the temporal and mechanistic relationships among B cell infiltration, gingival, and systemic inflammation, keeping in mind the phenotypic changes of T cells and macrophages in obesity-associated PD.

Our previous studies and new work herein showed that B cells from PD and T2D patients and obese, P. gingivalis-inoculated mice support production of TNF-α and/or IL-8/MIP-2 [11, 15]. These finding are important, as TNF-α is heavily implicated in unresolved oral inflammation and the resultant bone resorption [38, 39], and TNF-α antagonists reduce the development of inflammation and tissue injury in PD [40]. In addition to promoting osteoclastogenesis [41, 42], TNF-α may decrease tissue regeneration directly in PD by promoting fibroblast apoptosis [43]. Similarly, TNF-α plays a critical role in obesity-associated T2D, as TNF-α-null mice resisted insulin resistance following diet-induced obesity. Taken together, these findings suggest that B cell TNF-α production may be important in T2D-associated PD [44]. Furthermore, IL-8 is implicated in PD, T2D, and T2D-associated PD [11, 15, 45], but in contrast to the direct tissue damage orchestrated by TNF-α, IL-8 functions in PD through its ability to direct immune cell trafficking [22]. Although the current studies independently indicate that in vivo PD pathogen exposure in obesity primes B cells to promote proinflammatory cytokine production, and B cells from patients hyperproduce these PD-supporting cytokines, future studies will be required to determine whether B cells are the direct/sole source of TLR-downstream cytokines.

Overall, we conclude that B cells promote obesity-associated PD and oral pathogen-associated systemic inflammation through elevated proinflammatory cytokine secretion. In contrast, B cells play a minor (if any) role in PD pathogenesis in lean hosts. Thus, our data justify continued analysis of B cell function in human T2D-associated PD to begin to test the clinically important possibility that relatively safe B cell-depletion drugs, used for obesity and/or T2D treatment, may simultaneously alleviate PD.

Supplementary Material

Supplemental Data
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PRESS RELEASE
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ACKNOWLEDGMENTS

This study was funded by the U.S. National Institutes of Health (R21DK089270, 5R21DE021154, and R01DE15566), Hematology Training Program (HL007501), Boston Area Diabetes Endocrinology Research Center Pilot Program, Boston Nutrition Obesity Research Center (DK046200), and Evans Center for Interdisciplinary Biomedical Research Affinity Research Collaborative on Obesity, Cancer and Inflammation at Boston University.

The authors thank Drs. Frank Gibson, Ellen Weinberg, Basha Shaik-Dasthagirisaheb, and Nasi Huang for ongoing critiques and technical expertise. The Forsyth IHC Core and Joel Nikolajczyk provided expert technical assistance.

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

ABC
alveolar bone crest
AT
adipose tissue
CEJ
cementoenamel junction
CEJ-ABC distance
distance between the cementoenamel junction and alveolar bone crest
eAT
epididymal adipose tissue
Foxp3
forkhead box p3
GTT
glucose tolerance test
HFD
high-fat diet
IHC
immunohistochemistry
Klrb1c
killer cell lectin-like receptor subfamily B member 1C
LFD
low-fat diet
Pam3Csk4
palmitoyl-3-cysteine-serine-lysine-4
PD
periodontitis
RANKL
receptor activator of NF-κB ligand
Retnla
resistin-like α
T2D
type 2 diabetes
TRAP
tartrate-resistant acid phosphatase
WT
wild-type

AUTHORSHIP

M.Z. designed and carried out experiments, analyzed data, and wrote the manuscript. A.C.B. carried out flow cytometry experiments and analyzed data. J.D. carried out metabolic experiments and analyzed data. J.D.C. maintained mice and helped with metabolic experiments. T.E.V.D. was involved in experimental design, data analysis, and preparation of the manuscript. R.G. carried out experiments and was involved in experimental design, data analysis, and manuscript preparation. B.S.N. was involved in experimental design, data analysis, funding, compliance, and manuscript preparation.

DISCLOSURES

The authors declare no conflict of interest.

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