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

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.

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

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

(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).

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

(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.

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

(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.

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

(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.

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

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.

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

(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.

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

(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.