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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2008 Dec 31;85(4):648–655. doi: 10.1189/jlb.0708428

B cells from periodontal disease patients express surface Toll-like receptor 4

Hyunjin Shin *,1, Yue Zhang *,1, Madhumita Jagannathan †, Hatice Hasturk ‡, Alpdogan Kantarci ‡, Hongsheng Liu ‡, Thomas E Van Dyke ‡, Lisa M Ganley-Leal §,3, Barbara S Nikolajczyk *,2,3
PMCID: PMC2718806  PMID: 19118102

Abstract

Chronic systemic inflammation links periodontal disease (PD) to increased incidence of cardiovascular disease. Activation of TLRs, particularly TLR4, promotes chronic inflammation in PD by stimulating myeloid cells. B cells from healthy individuals are generally refractory to TLR4 agonists as a result of low surface TLR4 expression. Unexpectedly, a significantly increased percentage of gingival and peripheral blood B cells from patients with PD expressed surface TLR4. Surface expression correlated with an active TLR4 promoter that mimicked the TLR4 promoter in neutrophils. B cells from PD patients were surface myeloid differentiation protein 2-positive and also packaged the enhancer of a proinflammatory cytokine, IL-1β, into an active structure, demonstrating that these cells harbor key characteristics of proinflammatory cell types. Furthermore, B cells lacked activating signatures of a natural IL-1β inhibitor, IL-1 receptor antagonist. Surprisingly, despite multiple signatures of proinflammatory cells, freshly isolated B cells from PD patients had decreased expression of TLR pathway genes compared with B cells from healthy individuals. Decreases in inflammatory gene expression were even more dramatic in B cells stimulated with a TLR4 ligand from a periodontal pathogen, Porphyromonas gingivalis LPS 1690. In contrast, B cell TLR4 was not activated by the prototypic TLR4 ligand Escherichia coli LPS. These findings raise the unexpected possibility that TLR4 engagement modulates B cell activation in PD patients.

Keywords: human, bacterial infection, inflammation, gene regulation, molecular biology

INTRODUCTION

Chronic inflammation underlies multiple diseases, including periodontal disease (PD). Recent studies demonstrate that local inflammation in PD is mirrored by increased systemic inflammation that likely explains the link between PD and cardiovascular disease (CVD) [1,2,3]. The ability of oral pathogens such as Porphyromonas gingivalis to induce TLR4 expression and inflammatory protein production in aortic endothelial cells, indicates TLR4 plays a role in the relationship between PD and CVD [4]. TLR4 also plays a role in systemic inflammation via innate immune system cells that secrete proinflammatory cytokines into circulation in response to TLR4 ligands, including P. gingivalis [5, 6]. The demonstration that P. gingivalis is present in atherosclerotic plaques further highlights a role for TLR4/P. gingivalis interactions in the link among PD, systemic inflammation, and CVD [6].

An important, overall role for B cells in PD is suggested by demonstrations that the vast majority of cells in periodontal lesions (>65%) are B cells and plasma cells and that the proportion of B cells in these lesions correlates with disease severity [7,8,9]. B cells contribute directly to PD by producing the autoreactive antibodies identified in PD tissues and blood. These antibodies recognize collagen and other extracellular matrix proteins, likely contributing to local tissue destruction [10, 11]. Indirect roles for B cells in exacerbating PD are also likely. For example, the T cell costimulatory molecule CD86 is up-regulated in B cells within PD lesions [12], suggesting B cells influence T cell activity in PD. Berglundh and Donati [8] have also suggested a role for B cell antigen presentation in PD. Overall, B cells can play roles in local and systemic manifestations of PD, based on their ability to become activated by the chronic PD infection before recirculating throughout the body.

The link between TLR4 activity and PD led us to question whether B cell expression of TLR4 influences the B cell response to chronic PD infection. Mice express TLR4 on B cells, but recent studies show human B cells express little surface TLR4 [13,14,15,16]. However, TLR expression on human B cells may be altered under physiological conditions, raising the possibility that B cells from PD patients express TLR4 and therefore, respond to TLR4 ligands [14, 15, 17]. If in fact B cells from PD patients aberrantly express TLR4, B cells would have the potential to modify local and systemic inflammation as a result of their intrinsic ability to recirculate following activation.

We present data demonstrating an elevated percentage of peripheral blood and tissue B cells from PD patients express surface TLR4. B cells from PD patients also have signatures of a transcriptionally active IL-1β enhancer and are surface myeloid differentiation protein 2 (MD-2)-positive, suggesting their potential to alter the cytokine milieu during inflammation. Fresh ex vivo B cells from PD patients have altered expression of pro-inflammatory TLR pathway genes compared with B cells from healthy donors. Decreases in inflammatory gene expression and especially inflammatory transcription factor expression were more dramatic in B cells stimulated with a P. gingivalis LPS shown to be a monocyte-activating TLR4 ligand. Our findings support the interpretation that P. gingivalis LPS/TLR4 interaction leads to two fundamentally different responses by cells from PD patients: activation (of monocytes) versus inactivation (of B cells). Overall, these findings demonstrate that the proposed strategy of regulating systemic inflammatory disease by regulating TLR4 expression/activation [18] must account for this newly identified source of TLR4 activity, B cells.

MATERIALS AND METHODS

Cells

Human samples were obtained following informed consent under a Boston University (Boston, MA, USA) Institutional Review Board-approved protocol. Peripheral blood was collected into heparinized tubes by venous puncture from healthy volunteers or patients with a diagnosis of aggressive periodontitis (ref. [19]; herein designated PD) but no other known disease. The patients were characterized by periodontal infection with multiple organisms including P. gingivalis and Actinobacillus actinomycetemcomitans. In most cases, chronic infection leads to severe, aggressive, and early-onset bone loss around first molars and incisor teeth only. Clinical and radiographic criteria of PD included age of onset around the circumpubertal period (<13 years old) and alveolar bone loss localized around the first permanent molars and incisors [19]. In addition, the subject’s periodontal diagnosis was confirmed further by neutrophil functional analysis [20]. Systemically, healthy donors who had no sign of PD other than mild gingivitis were matched to PD patients based on age and sex when possible. Patients and healthy donors ranged from 17 to 52 years or 23 to 58 years, respectively. None of the subjects were smokers, and all PD and healthy donors were of African-American origin. Gingival tissue was from periodontal surgery patients with aggressive periodontitis. Noninflamed gingival tissue yielded too few B cells for analysis. Monocytes were purified from source leukocytes (NY Biologics, New York, NY, USA). Tonsils and appendices from the National Disease Research Interchange (Philadelphia, PA, USA) were processed as published [17]. Neutrophils from healthy donors were isolated from PBMCs on a histopaque-1077 gradient. B cells from healthy donors or PD patients were isolated from PBMCs using negatively selecting magnetic beads (Dynal, Norway) and were >99% pure, as analyzed by surface expression of lineage-specific markers, including CD19. B cells were rested for 1 h postpurification prior to further analysis. Mono-Mac-6 monocytes were cultured as described [21].

Flow cytometry

Whole blood (100 μl) was incubated with fluorescently labeled antibodies for 30 min at 4°C. Antibodies to the following antigens were purchased from BD PharMingen (San Diego, CA, USA): CD3, CD14, CD19, CD11b, and IgG1-FITC. α-TLR4 and α-MD-2 were purchased from eBioscience (San Diego, CA, USA). RBC were then lysed with 2 ml 1× FACS™ lysing solution (BD PharMingen) for 30 min at room temperature. Cells were washed with 0.2% BSA/PBS and resuspended in PBS. Purified B cells were labeled using standard procedures [17]. Data were collected on a FACScan (BD Biosciences, San Jose, CA, USA) using CellQuest software and analyzed with WinMDI software (Joseph Trotter, The Scripps Institute, Palo Alto, CA, USA).

Biochemistry

Chromatin accessibility by real-time PCR (CHART-PCR) and chromatin immunoprecipitation (ChIP) were analyzed as published [21] or with TLR4-specific primers: 5′-GCTAAGGTTGCCGCTTTCAC-3′ and 5′-CTTCCTCGAGCCGCCC-3′. The IL-1 receptor antagonist (IL-1ra)-specific primers were 5′-GGTATTTCCGCTTCTCGCAG-3′ and 5′-ACTCACCCAAGCTAGGCGTC-3′. ChIP antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were: α-histidine, α-cJun, α-PU.1, and α-IFN regulatory factor 8 (IRF-8); α-IRF-4 was from Dr. Michael Atchison (University of Pennsylvania, Philadelphia, PA, USA). IL-1β mRNA was quantified by real-time PCR as published [22], using β2-microglobulin as the normalization control and the ΔΔ-comparative threshold (Ct) method. Monocytes were stimulated for 4–20 h prior to mRNA analysis. Purified B cells were incubated on ice for 1 h prior to analysis on Superarray TLR signaling plates. One hour is a sufficient amount of time for cells to lose the low level of serine phosphorylation induced by the negatively selecting magnetic bead procedure (not shown). Alternatively, isolated B cells were stimulated with highly purified P. gingivalis 1690 LPS ± P. gingivalis 1435/1449 LPS for 24 h before Superarray analyses.

Statistical analyses

The Mann-Whitney U or Kruskal-Wallis test was used for nonparametric comparisons of group means for B cells from peripheral blood or tissues, respectively (GraphPad InStat, San Diego, CA, USA). ChIP and Superarray results were analyzed by Student’s t-test.

RESULTS

The link among TLR4, PD, and CVD led us to question whether TLR4 expression changes within the proinflammatory milieu of PD patients. We quantified surface TLR4 on B cells from gingival tissues or blood from PD patients or healthy donors. We found an average of 26.0% of CD19+ B cells from PD blood and 55.6% of B cells from PD gingiva expresses TLR4 (Fig. 1, A–C). In contrast, an average of 12.1% of peripheral blood B cells from healthy donors and few peripheral blood T cells from PD patients expressed TLR4 (Figs. 1, A and B, and 2A). Multiple attempts at analyzing B cells from healthy gingival tissue failed to recover a measurable number of B cells (not shown). Most circulating B cells (>95%) from both cohorts expressed the receptor RP105 (not shown). Next, we questioned whether TLR4 expression is unique to B cells from PD patients. B cells isolated from two distinct inflammatory sites, inflamed appendix and tonsil, failed to express TLR4 significantly (Fig. 1C). We conclude that PD, but not inflammation per se, provides the appropriate milieu for TLR4 up-regulation on B cells.

Fig. 1.

Fig. 1.

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TLR4 is up-regulated on B cells from patients with PD. Fresh whole blood or isolated B cells from tonsillitis tonsils, appendicitis appendices, or PD gingiva were analyzed by flow cytometry for surface TLR4 expression. (A) Representative plots from one blood sample from a PD patient or healthy donor or gingival B cells from a PD patient as indicated. Boxes indicate the percentage of B cells that expresses TLR4 and were drawn using isotype control as background (right panel for gingival tissue, and data not shown for whole blood). (B) Composite data showing percent TLR4+ B cells in the peripheral blood of healthy (left; n=19) or PD (right; n=17) donors. The two cohorts have significantly different percentages of TLR4+ B cells in circulation (*, P=0.0074, by nonparametric Mann Whitney U test). (C) Composite data showing percentage of TLR4+ B cells in tonsillitis (n=10), appendicitis (n=5), and PD gingival tissue (n=7). Healthy gingival tissue was not available for analysis. The difference in TLR4 expression on B cells from PD tissues versus tonsil or appendix is highly significant (*, P=0.001, by the Kruskal-Wallis test). Percentage of TLR4+ B cells is similar between tonsil and appendix (P>0.05).

Fig. 2.

Fig. 2.

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B cells from PD patients have molecular signatures of TLR4 activation. (A) TLR4 expression on CD3+ T cells from a representative PD patient of the three patients analyzed. (B) A representative plot of purified B cells used for biochemical analyses of the TLR4 gene in panels C and D. B cells were routinely devoid of significant contamination with monocytes, as shown, and CD3+ T cells (not shown). FACS plot is representative of multiple, independent B cell purifications. In no case were B cells less than 98% pure. (C) The TLR4 promoter of purified monocytes or B cells from healthy or PD patients was treated with the indicated units of MNase prior to analysis by CHART-PCR. Slightly lower accessibility in B cells from PD patients versus healthy donors is unlikely to be biologically significant compared with the complete inaccessibility of the promoter in human embryonic kidney (HEK) 293 cells and similar accessibility in monocytes. Results show averages and range of three independent determinations. (D) Analysis of TLR4/transcription factor binding in B cells from PD patients by ChIP using antibodies recognizing proteins as indicated. We conservatively designate twofold enrichment as an indication of association. his, Histidine. *, Significant difference (P<0.05 by Student’s t-test). (E) Analysis of TLR4/transcription factor binding in neutrophils from healthy donors by ChIP using antibodies recognizing proteins as indicated. (D) Average and se of four to eight independent determinations. (E) Average and range of two independent determinations.

To verify TLR4 expression independently, we analyzed molecular signatures of an active TLR4 gene in highly purified B cells (Fig. 2B). The TLR4 promoter is packaged into an inaccessible chromatin structure in HEK 293 cells (Fig. 2C). In contrast, the TLR4 promoter in blood B cells from healthy donors or PD patients was MNase-accessible (i.e., nucleosome-depleted), similar to the monocyte promoter (Fig. 2C). The potential of a higher percentage of MNase-accessible TLR4 loci in B cells from healthy versus PD donors at lower enzyme concentrations is unlikely to be biologically significant, given the quantitatively similar levels of TLR4 promoter accessibility in B cells from PD patients versus monocytes known to express the TLR4 gene and the high variability among PD samples assayed at lower enzyme concentrations. Although these findings show that B cells and monocytes package the TLR4 promoter into an “active” chromatin structure, they do not explain why a higher percentage of B cells from PD patients expresses surface TLR4.

As chromatin structure did not differentiate B cells from PD patients versus healthy donors, we analyzed direct association of known TLR4 activators with the TLR4 gene. Monocyte TLR4 transcription is regulated by PU.1 and IRF-4/8 [23, 24]. The TLR4 promoter also contains an AP-1 (c-Jun/fos) element [24]. Peripheral blood B cells from healthy donors lacked transcription factor association with the TLR4 promoter (Fig. 2D, black bars), but B cells from PD patients had significant PU.1/TLR4 promoter association (*). cJun and IRF association with the TLR4 promoter was similar between B cells from PD patients and healthy donors. This pattern of transcription factor association recapitulated associations found in TLR4-expressing, unstimulated neutrophils from healthy donors (Fig. 2E). Taken together, findings in Figures 1 and 2 raised the possibility of a new modulator of inflammation in PD patients, TLR4+ B cells.

To further understand the breadth of changes in B cells from PD patients, we measured additional signatures generally associated with pro-inflammatory cell types. First, we measured B cells for the presence of surface MD-2, a receptor that appears to form a bridge between TLR4 and its ligands [25]. Figure 3A shows that B cells from PD patients are surface MD-2+ and suggests that B cell TLR4 is functional. Next, we focused our analyses on activation of a second key pro-inflammatory gene in innate immune system cells, IL-1β, focusing on transcription factor/gene associations implicated in IL-1β induction [21, 26,27,28]. IRF-4 constitutively associated with the IL-1β enhancer in B cells from PD patients but not healthy donors (Fig. 3B), indicating an activated IL-1β gene. However, unlike monocytes, B cells from PD and healthy donors lacked constitutive transcription factor association with the IL-1β promoter (Fig. 3C and refs. [21, 22]). Furthermore, B cells from PD patients and healthy donors package the IL-1β promoter into a minimally accessible chromatin structure that contrasts with the highly accessible monocyte IL-1β promoter (Fig. 3D). These promoter analyses explain, at least in part, why B cells from PD patients (and B cells in general) fail to constitutively produce IL-1β as analyzed by ELISA (data not shown). However, these findings raise the possibility that appropriate stimulation may activate B cells to produce IL-1β under some circumstances. Similarly, B cells from PD patients do not appear to constitutively activate the natural inhibitor of IL-1β, IL-1ra, as evidenced by the lack of PU.1/IL-1ra promoter association (Fig. 3E) thought to play a role in IL-1ra activation [29]. Measurement of in vivo PU.1/IL-1ra promoter association in neutrophils (Fig. 3F) confirmed previous studies, suggesting that PU.1 regulates IL-1ra and serve as a positive control [29, 30]. Overall, these findings are consistent with our hypothesis that B cells in PD patients but not healthy donors harbor some but not all signatures of pro-inflammatory cells.

Fig. 3.

Fig. 3.

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B cells from PD patients have additional signatures of cells that modulate inflammation. (A) MD-2, the protein that bridges TLR4 with ligand [25], is detectable on the surface of TLR4+ B cells, as analyzed by flow cytometry. Virtually all B cells in PD patients that were TLR4+ were also MD-2+. Shown is a representative of three independent analyses. (B) ChIP assays show transcription factor association with the IL-1β enhancer in B cells from the indicated donors (x-axis). IRF-4 does not associate with an unrelated region of the IL-1β gene that lacks a consensus-binding sequence (not shown). *, Differences in IRF-4/enhancer association between B cells from PD versus healthy donors are highlighted (P<0.05 by Student’s t-test). (C) Transcription factor association with the IL-1β promoter in B cells from PD patients. Bars are averages and se of greater than or equal to three independent determinations for B and C. (D) IL-1β promoter accessibility to MNase (2 U) in cell types indicated above each bar. Shown are average and range of greater than or equal to two independent determinations. (E and F) Association between the transcription factor PU.1 and the natural competitive inhibitor of IL-1β, IL-ra, in (E) B cells from PD patients or (F) neutrophils from healthy donors. Shown are averages and ranges of two to three independent determinations from the same ChIP samples amplified in B and C and Figure 2, D and E.

We reasoned that because the pro-inflammatory milieu of PD patients correlates with TLR4 expression and that TLR4 ligands are copious in the chronically infected PD gingiva, the patient environment may provide TLR4 ligands to constitutively activate TLR4. To test this possibility, we isolated B cells from peripheral blood of PD patients or healthy donors and rested them for 1 h to neutralize cellular activation as a result of the isolation procedure. We then analyzed constitutive TLR pathway activation on Superarray plates. Multiple TLR target genes were up- or down-regulated more than twofold in B cells from each of three PD patients as compared with each of three healthy donors (a total of nine comparisons). Three transcription factors, c-fos, c-Jun (i.e., AP-1), and IRF-1 were constitutively up-regulated an average of five- to eightfold in B cells from PD patients (Fig. 4A, black bars). The NF-κB proteins RelA and cRel were up-regulated modestly (∼2×; data not shown). However, IFN-β was hypoexpressed significantly in B cells from PD patients (white bar). IFN-α, -γ and IL-10 were expressed in B cells from healthy donors but were not expressed in B cells from PD patients. Calculations could not be made for this comparison as a result of a lack of Ct for the PD samples. These findings are consistent with the possibility that these cells, despite having increased TLR4 and AP-1 levels and a protein-associated IL-1β enhancer (Fig. 3B), have encountered an activation-blocking ligand in vivo.

Fig. 4.

Fig. 4.

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B cells from PD patients constitutively up- and down-regulate genes in TLR pathways. (A) Superarray plate analysis comparing unstimulated B cells from three PD patients and three healthy donors (for a total of nine comparisons). Shown are genes up-regulated (black bars), unchanged (gray bars), or down-regulated (white bar) in B cells isolated from PD peripheral blood, purified and then rested for 1 h on ice prior to analysis. (B) Gene expression in B cells from PD patients versus healthy donors incubated ex vivo in media alone for 24 h. Shown is the average and sd of values obtained from two donor samples from each cohort (for a total of four comparisons). *, Statistical significance (P<0.05) by the Student’s t-test. Additional genes that approached significant differences (P<0.08) were IRF-1 and cJun (A) and MyD88 and NF-κB-1 (B). IKK-beta, IκB kinase; AGMX1, Bruton’s tyrosine kinase.

A similar analysis of B cells incubated for 24 h in the absence of ex vivo stimulation demonstrated that in general, differences in TLR target gene expression between B cells from PD patients versus healthy donors were qualitatively and quantitatively different from results with fresh ex vivo B cells (compare Fig. 4, A and B). Increased c-fos, c-Jun, and IRF-1 were not maintained outside the in vivo environment, and two of these three genes had significantly decreased expression levels in B cells from PD patients versus healthy donors (Fig. 4B; *, P<0.05). Other genes, such as IL-6, were similarly expressed in fresh ex vivo and in vitro-incubated B cells from patients or healthy donors, indicating that baseline expression of some genes is at least somewhat independent of external stimuli. Decreased expression of other genes, such as MyD88 and NF-κB1, approached statistical significance (P=0.07 or 0.08, respectively) only in B cells from PD patients incubated in media for 24 h (Fig. 4B). These findings suggest that changes in TLR pathway activation in B cells from PD patients versus healthy donors shown in Figure 4A may require continuous input from the PD environment. The likelihood that TLR pathway genes are activated through other surface receptors on B cells, including other TLRs, could not be dismissed by these analyses.

We next tested the possibility that B cell TLRs, perhaps in combination with the in vivo TLR4 ligands suggested by results in Figure 4, influence the inflammatory potential of B cells. We first measured the response of B cells from PD patients and healthy donors to the prototypic TLR4 ligand, Escherichia coli LPS. Surprisingly, all B cells, regardless of surface TLR4 expression, failed to respond to E. coli LPS as measured by IκB-α degradation (Fig. 5, A and B) and TLR pathway array analyses (data not shown). This finding contrasts with our demonstration of IκB-α degradation and resynthesis over the same time course in our previous work on monocytes [22]. This finding could be explained by two independent interpretations: B cell TLR4 is nonresponsive to E. coli LPS, or B cell TLR4 toleration in vivo blocks the response completely.

Fig. 5.

Fig. 5.

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Cellular responses to TLR4 ligands, as measured by changes in gene expression and NF-κB pathway activation downstream of TLR. (A and B) Western blots quantifying IκB-α levels in B cells from PD patients (A) or healthy donors (B), treated as indicated. E. coli LPS was added at 1 μg/ml. The proteosome inhibitor MG132 blocks IκB-α degradation in activated cells. Percentage of TLR4+ B cells was 39.3% or 15.1% for samples from PD or healthy donors, respectively. Caspase-1 (Casp-1) was the loading control (lower panels). Results are representative of three independent analyses. (C and D) TLR signaling Superarray analysis of B cells from PD patients (white bars) or healthy donors (black bars) incubated with the TLR4-activating P. gingivalis LPS 1690 [31] (C, 10 μg/ml for 24 h) versus media alone or P. gingivalis LPS 1690 versus a combination of P. gingivalis LPS 1690 + P. gingivalis LPS 1435/1449 [31] (D, 10 μg/ml each for 24 h). Genes are arranged as in Figure 4A for comparison. Average and range of results from two donor samples are shown. Negative values indicate that mRNA levels in P. gingivalis LPS 1690-treated cells are lower than mRNA levels in cells treated with (C) media or (D) P. gingivalis LPS 1690 + P. gingivalis LPS 1435/1449. Genes whose expression was variably changed by the TLR4 ligand are not shown. A twofold difference was defined as a change in gene expression in these assays, as indicated by the dotted lines. (E) Monocyte response to P. gingivalis LPS 1690. Mono-Mac-6 human monocytes were stimulated with 10 ng/ml E. coli LPS or 10 μg/ml P. gingivalis LPS 1690 ± P. gingivalis LPS 1435/1449 for 4 h as indicated by the key. Monocytes were then analyzed for IL-1β mRNA levels by quantitative PCR. Shown are average and sd of three independent determinations.

To test whether TLR4+ B cells fail to respond to E. coli LPS as a result of tolerance, an otherwise functionally inactive receptor, or an unexpected activity of TLR4, we took advantage of the demonstration that different TLR4 ligands can induce different downstream responses in monocytes. We tested the possibility that in contrast to E. coli LPS, a TLR4 ligand from a periodontal pathogen, P. gingivalis LPS, might elicit a response in B cells from PD patients. We measured cellular responses to two LPS preparations from P. gingivalis: 1690, which activates cells through MD-2/TLR4, and P. gingivalis 1435/1449, which activates cells through TLR4 or TLR2. P. gingivalis 1435/1449 can act as a weak agonist or an antagonist of P. gingivalis 1690 [31, 32]. In contrast to monocytes, TLR4-activated genes have not been identified for human B cells. We therefore measured the global response of B cells from PD patients to P. gingivalis LPS on TLR pathway array plates. B cells from PD patients treated with P. gingivalis 1690 had decreased expression of multiple TLR target genes compared with B cells incubated in media alone (Fig. 5C, white bars). Interestingly, P. gingivalis LPS 1690 treatment decreased expression of the pro-inflammatory transcription factors IRF-1 and c-Jun in B cells from PD patients but not healthy donors. Decreased gene expression in P. gingivalis LPS-treated B cells contrasts with the response to the TLR4 ligand in TLR-tolerated cells that would, according to the definition of tolerized, be greater than or equal to gene expression in untreated cells. Changes in gene expression by B cells from healthy donors incubated in P. gingivalis 1690 versus media were more modest (Fig. 5C, black bars). A small population of TLR4+ B cells in healthy individuals may, for example, explain the IL-6 up-regulation in response to P. gingivalis 1690. P. gingivalis LPS failed to activate the IL-1β transcript in all B cells (not shown), contrasting with activation demonstrated in monocytes (Fig. 5E). Overall, these results demonstrated that a TLR4 ligand from a PD pathogen decreases activation of TLR pathway genes in B cells from PD patients but not healthy donors.

To further test differences in the B cell versus monocyte responses to TLR4 ligands from an oral pathogen, we incubated cells from PD or healthy donors with a combination of two oral pathogen TLR4 ligands, P. gingivalis LPS 1690 + P. gingivalis LPS 1435/1449 (Fig. 5D). This combination of P. gingivalis LPSs activated inflammatory gene expression in monocytes additively (Fig. 5E, white bar). B cells from PD patients but not healthy donors treated with P. gingivalis 1690 alone had significantly decreased amounts of mRNA for IRF-1 and c-Jun expression compared with cells costimulated with P. gingivalis 1690 + P. gingivalis 1435/1449 (Fig. 5D, white bars, P<0.02). These data support the conclusion that P. gingivalis 1435/1449 blocks the ability of P. gingivalis 1690 to decrease expression of some TLR pathway gene expression. This result reinforces the antagonistic properties of P. gingivalis LPS 1435/1449 versus LPS 1690 described previously [32], although for B cells, LPS 1435/1449 blocks a negative response to LPS 1690. The blocking effect of P. gingivalis LPS 1435/1449 on 1690 function on B cells contrasts further with the additive function of these LPS in monocyte gene activation (Fig. 5E). These results therefore represent an example of cell type-specific TLR responses to the same ligand(s). Dual P. gingivalis LPS stimulation had little effect on the TLR pathway gene expression in B cells from healthy donors (Fig. 5D, black bars). Taken together, the B cell gene expression in response to P. gingivalis and E. coli LPS support the likelihood that the B cell response to multiple TLR4 ligands is substantially different from the monocyte response. Our data further support the unanticipated conclusion that TLR4 on B cells from PD patients responds to TLR4 ligands from oral pathogens predominantly by decreasing pro-inflammatory gene expression. Furthermore, B cells from healthy donors fail to respond significantly to TLR4 ligands. These findings highlight the likely anti-inflammatory/anti-activation function for TLR4 on human B cells.

DISCUSSION

Our results show that an increased percentage of B cells from PD patients expresses surface TLR4. Mechanisms underlying TLR4 up-regulation in B cells are unknown but may be influenced by the microbiota, as suggested by up-regulation of TLR4 on B cells in patients with oral infections but not tonsillitis or appendicitis. Regardless, B cells from PD patients cannot be characterized as simply pro-inflammatory or anti-inflammatory relative to cells from healthy donors. For example, the “pro-inflammatory” transcription factors, such as c-fos and c-Jun, which are constitutively up-regulated in B cells from PD patients, may be balanced by decreased IFN-β activation in B cells from PD patients. This finding replicates results in human macrophages, where AP-1 (fos/Jun heterodimer) is up-regulated in the absence of increased IFN-γ, despite the demonstration that AP-1 can activate IFN-γ in some contexts [33]. Taken together, these findings support the suggestion that AP-1 does not function exclusively as a pro-inflammatory transcription factor [33]. The demonstration that P. gingivalis LPS 1690 blocks rather than activates transcription factor up-regulation highlights the likelihood that the microbial ligands putatively responsible for altering the phenotype of freshly isolated B cells in vivo are somewhat different or more complex from our in vitro stimulants. Oral pathogens likely activate multiple TLRs and other receptors in combination to produce different stimulation outcomes in vivo. Regardless, the ability of P. gingivalis LPS to decrease multiple inflammatory signatures relative to B cells cultured in the absence of stimulation supports the clinically important possibility that B cells in PD patients can be inactivated through exogenous TLR4 ligands, despite the inevitable presence of in vivo stimulants. Importantly, our demonstration that inflammatory gene expression decreases in B cells from PD patients but not healthy donors under standard tissue-culture conditions highlights the role that continuous exposure to bacterial ligands may play in altering B cell phenotype in PD patients. These results further suggest caution in extrapolating results from relatively long-term ex vivo B cell cultures to likely in vivo B cell functions in the absence of corroborating experiments in fresh ex vivo B cells. Overall, we conclude that there are profound changes in circulating B cells from PD patients, at least some of which appear to reflect an in vivo encounter with the inflammatory milieu of PD.

One possible explanation for the lack of a positive B cell response to P. gingivalis LPS TLR4 ligands is that B cells in PD patients are tolerized to TLR-mediated activation by in vivo interaction with other TLR ligands. TLR homo- or heterotolerance, defined by a less robust (or no) response to subsequent challenge with TLR ligands, may explain why B cells from PD patients fail to respond to E. coli LPS. In contrast to the lack of response characterizing a TLR-tolerized cell, B cells from PD patients respond to TLR4 ligands by decreasing gene expression below fresh ex vivo mRNA levels for most genes tested, both relative to B cells from healthy donors. Therefore, instead of a blunted, “induced tolerance” response, the data support the existence of a mechanism, previously unappreciated, which decreases gene expression below steady-state in vivo levels in B cells from PD patients.

The surprising finding that B cells up-regulate TLR4 expression in PD patients led us to investigate the molecular mechanisms of TLR4 activation in these cells. The TLR4 promoter is nucleosome-free in B cells from healthy donors (Fig. 2C), in contrast to the highly inaccessible promoter structure assumed in, for example, HEK 293 cells. These findings show that B cells from healthy individuals may be poised to activate TLR4 expression following stimulatory events that would be unable to activate the gene in other cells types. This interpretation is supported by the demonstration that T cells from PD patients are surface TLR4-negative (Fig. 2A), despite being exposed to the same systemic, inflammatory environment. Similarly, peripheral blood monocytes from inflammatory disease patients versus healthy donors express identical levels of TLR4 (not shown), suggesting increased TLR4 expression is limited to B cells. Although PU.1, IRF-4, and IRF-8 are constitutively expressed in normal B cells [34,35,36], and the TLR4 promoter is constitutively accessible, these transcription factors do not associate with the TLR4 promoter (Fig. 2D). The mechanism limiting association of these proteins with an accessible TLR4 promoter in B cells is unknown, but these findings show that mechanisms regulating TLR4 transcription are incompletely understood. As PU.1 expression levels do not change in response to stimulation (at least in monocytes), we predict that PU.1 phosphorylation, along with IRF protein phosphorylation [37], may be important for activating the TLR4 gene in B cells from PD patients.

The biological significance of partial IL-1β gene activation in B cells from PD patients is unknown. We were unable to stimulate IL-1β mRNA or protein production from B cells by various stimulation protocols, including E. coli LPS, alone or in combination with α-Igμ and α-CD40 (not shown). We speculate that ongoing characterization of B cells from PD and other systemic inflammatory disease patients will identify up-regulation of additional receptors that predict ligands to drive IL-1β transcription. Based on the important role IL-1β plays in PD and other systemic inflammatory diseases, a contribution of B cells to the net pool of IL-1β would further change the way clinicians think about targeting pro-inflammatory cell types toward interrupting the link between PD and, for example, CVD.

Acknowledgments

This work was supported by R01 AI54611 and a research grant from the American Diabetes Association (B. S. N.), the Evans Medical Foundation (L. M. G-L.), and USPHS grant DE15566 (T. E. V. D.). We thank YanMei Liang for technical support and Tom Chiles and Michael Clare-Salzler for manuscript critiques. Frank Gibson provided technical information about P. gingivalis LPS.

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