molecular oral microbiology molecular oral microbiology
Periodontitis in the absence of B cells and specific anti-bacterial antibody J. Oliver-Bell1, J.P. Butcher2, J. Malcolm1, M.K.L. MacLeod1, A. Adrados Planell1, L. Campbell1, R.J.B. Nibbs1, P. Garside1, I.B. McInnes1 and S. Culshaw3 1 Institute of Infection, Immunology and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 2 Institute of Biomedical and Environmental Health Research, School of Science, University of the West of Scotland, Paisley, UK 3 Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
Correspondence: Shauna Culshaw, Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, UK Tel.: + 44 141 211 9733; E-mail: [email protected] Keywords: B cells; murine models; periodontitis Accepted 7 September 2014 DOI: 10.1111/omi.12082
SUMMARY Periodontitis (PD) results from complex interactions between a dysbiotic oral microbiota and a dysregulated host immune response. The inflammatory infiltrate in the gingiva of PD patients includes an abundance of B cells, implicating these cells in the immunopathology. We sought to investigate the role of B cells in PD using a murine model. Wild-type or B-cell-deficient (lMT) mice were orally infected with Porphyromonas gingivalis. One or six weeks following infection, lymphocyte populations in the gingiva and cervical draining lymph nodes (dLN) were analysed by flow cytometry; serum anti-P. gingivalis IgG antibody titers were measured by enzyme-linked immunosorbent assay, and alveolar bone loss was determined. In wild-type mice, the percentage of gingival B cells expressing receptor activator of nuclear factor-jB ligand (RANKL) was significantly increased 1 week post-infection (5.36% control versus 11% PD, P < 0.01). The percentage of Fas+ GL7+ germinal centre B cells in the dLN was significantly increased at both 1 week (2.03% control versus 6.90% PD, P < 0.01) and 6 weeks (4.45% control versus 8.77% PD, P < 0.05) postinfection. B-cell-deficient mice were protected from P. gingivalis-induced alveolar bone loss, 160
with a lack of B-cell proliferation and lack of CD4+ CD44+ CD62L T-cell generation in the dLN, and absence of serum anti-P. gingivalis antibodies. Our data imply a pathological role for B cells in PD, and that selective targeting of this immune axis may have a role in treating severe periodontal disease.
INTRODUCTION The inflammatory infiltrate in gingival tissue in periodontitis (PD) includes an abundance of B cells, along with their survival factors interleukin-6, A proliferation inducing ligand (APRIL) and B-cell activating factor (BAFF) (Gumus et al., 2014). Many studies have reported that plasma cells account for half of the leukocyte infiltrate, and B cells for a further 20% (Berglundh & Donati, 2005) In contrast, there are relatively few leukocytes in healthy gingivae, of which only about 5% are B cells (Gemmell et al., 2002). Tissue-resident plasma cells in patients with PD secrete antibodies of immunoglobulin G (IgG), IgA and IgM classes (Mackler et al., 1977; Seymour & Greenspan, 1979), including antibodies specific for bacteria associated with PD, including Porphyromon© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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as gingivalis (Apatzidou et al., 2005). Locally produced anti-P. gingivalis antibodies may contribute to the elevated titers detectable in the saliva and serum of patients. However, it is unclear whether these antiP. gingivalis antibodies are protective, destructive or irrelevant to disease status. Intriguingly, these antibodies appear more frequently in the serum of patients with concurrent rheumatoid arthritis, and may form part of the link between PD and rheumatoid arthritis (Kaur et al., 2013). Studies of PD patients before and after periodontal treatment, or studies comparing PD patients with healthy patients have consistently demonstrated a specific antibody response, but the role of that antibody remains difficult to define (Gmur et al., 1986; Takeuchi et al., 2006; Okada et al., 2013). The generally unresolving nature of chronic PD would imply that the on-going antibody production in a patient with disease is ineffective or insufficient in managing the oral microbiome. Moreover, the anti-bacterial humoral response is often accompanied by increased levels of local and circulating autoantibodies reactive to citrullinated peptides (Harvey et al., 2013; Lappin et al., 2013; de Pablo et al., 2014) and collagen (Hirsch et al., 1988). These autoantibodies may exacerbate tissue destruction and bone loss through the formation of immune complexes and the stimulation of Fc receptor bearing immune cells. The anti-citrulline autoantibodies found in patients with PD are hypothesized to offer some explanation of the link between PD and rheumatoid arthritis, the latter strongly associated with such antibodies (Klareskog et al., 2008). In addition to the generation of an inappropriate auto-antibody response, atypical activation of B cells could contribute to PD pathogenesis by their antigen-presentation, cytokine production, and expression and secretion of receptor activator of nuclear factor-jB ligand (RANKL), the latter promoting osteoclastogenesis via its receptor, RANK (Yasuda et al., 1999). RANKL is reported to be expressed predominantly by lymphocytes in the gingiva of PD patients (Kawai et al., 2006; Wara-Aswapati et al., 2007). A key role for RANKL has been documented in rodent models of PD (Yuan et al., 2011; Han et al., 2013), and in vitro studies indicate increased lymphocyte expression of RANKL following culture with Aggregatibacter actinomycetemcomitans or P. gingivalis (Han et al., 2009; Belibasakis et al., 2011). The requirement for antigen specificity in this system was demonstrated © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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in vivo by adoptive transfer of antigen-experienced B cells specific for A. actinomycetemcomitans, which exacerbated RANKL-dependent alveolar bone loss in rats immunized with the same bacteria (Han et al., 2006, 2009). In addition to RANKL expression, B cells have myriad other functions that may perpetuate disease. Compared with conventional ‘B2’ B cells, the ‘B1’ B cell subset possesses particular autoreactive and host-damaging potential. B1 cells are more likely to escape central tolerance, and are less dependent on help from T cells to become activated (Fagarasan et al., 2000; Cao et al., 2011). Small clinical studies have suggested recruitment or proliferation of B1 cells in the gingiva and circulation of PD patients (Berglundh et al., 2002; Donati et al., 2009). This role of B cells can be directly probed using murine models of B-cell abnormality and deficiency. We have investigated changes in the expression of RANKL and other cell surface markers by B cells in the gingiva and draining lymph node (dLN) of normal mice following infection with P. gingivalis, and investigated the role of mature B cells in PD using B-cell-deficient lMT mice. Our findings lend support to the hypothesis that B cells are activated, are a source of RANKL, and seem to play a predominantly pathological role in PD. METHODS Murine model of periodontitis Female BALB/c (Harlan, Bicester, UK), C57BL/6 (Charles River, Kent, UK), or lMT mice [bred in house on a C57BL/6 background; (Kitamura et al., 1991)] were maintained before and during experiments in specific pathogen-free conditions with ad libitum access to food and water at Biological Services, University of Glasgow. All work was performed under licence in accordance with UK Home Office regulations and after ethical approval from local research ethics committees. Porphyromonas gingivalis W83 (ATCC, Middlesex, UK) was prepared as previously described and mice were orally infected with 109 colony-forming units P. gingivalis W83 in 2% carboxymethylcellulose (CMC) by gavage on 3 or 4 consecutive days as previously described (Baker et al., 1994). Control mice received CMC alone. At 1 or 6 weeks post-infection, serum, maxillary teeth, 161
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gingiva and dLN were collected following terminal general anaesthesia and cardiac puncture.
Antibody titers were calculated as described previously (Gmur et al., 1986).
Flow cytometry
Assessment of alveolar bone loss
Gingival tissue was dissected from the maxillae – encompassing the palatal mucosa, and a very thin strip of buccal tissue obtained from the buccal surfaces. Tissue was collected into 1 ml phosphatebuffered saline, incubated with 500 lg Liberase DH (Roche, West Sussex, UK) at 37°C for 30 min, then 2 ml cRPMI (RPMI-1640 with 10% fetal calf serum, 1% penicillin streptomycin, 1% L-glutamine; Gibco, Invitrogen, Carlsbad, CA, USA) was added and tissue was dissociated using gentleMACS C tubes (Miltenyi Biotec, Surrey, UK) cellular dissociation protocol, resulting in between 2 9 105 and 5 9 105 viable cells per mouse assessed by trypan blue exclusion. In addition, cell suspensions were obtained from four cervical dLN from each mouse. For flow cytometry, cell suspensions were resuspended in Fc block (5% mouse serum in 2.4G2 hybridoma supernatant) then fluorochrome-labeled antibodies against CD45, CD95 (eBioscience, Hatfield, UK), CD19, CD43, CD23, CD5, RANKL, CD138, CD3, GL7 (BD Biosciences, San Jose, CA, USA) or similarly labeled isotype control antibodies. Cells were analysed using a MACS Quant (Miltenyi Biotec), and data were analysed using FLOWJO (Tree Star Inc., Ashland, OR, USA).
The maxilla was separated from the skull and gingivae were removed. Maxilla were defleshed and treated as previously described (Baker et al., 1994). Images were captured using an Olympus SZX7 stereo zoom microscope fitted with SC100 digital color camera. Measurements of the distance between the cemento–enamel junction and the alveolar bone crest were made using IMAGEJ software (National Institute of Health, Bethesda, MD, USA) to assess alveolar bone loss as previously described (Baker et al., 1994).
Enzyme-linked immunosorbent assays Interleukin-6 and tumour necrosis factor-a enzymelinked immunosorbent assays (ELISAs; ReadySET-Go! Kit, eBioscience, Hatfield, UK) and sRANKL ELISAs (DuoSet kit, R&D Systems, Abingdon, UK) were performed according to the manufacturer’s instructions. Antibody titers to P. gingivalis in the serum samples were ascertained as previously described (Mooney et al., 1993) with minor modification. In brief, Dynotech Immunolon 1B microtiter plates (ThermoFisher, Loughborough, UK) were coated overnight with heat-killed P. gingivalis W83 at an OD600 0.02 in 100 mM bicarbonate buffer pH 9.6. After blocking, serum dilutions in phosphate-buffered saline were added then bound antibody was detected with biotin-labeled anti-mouse-IgG antibodies, extravidin-horseradish peroxidase and 3,3’,5,5’-tetrameth ylbenzidine substrate (all from Sigma, Poole, UK). 162
Statistics Data were analysed by Student’s t-test or analysis of variance with Tukey comparison, as indicated in the figure legends, using GRAPHPAD PRISM 5 (La Jolla, CA, USA). RESULTS To evaluate changes in local B-cell populations during initial and later stages of the murine oral infection model of PD, cells were isolated from the gingiva of mice at 1 and 6 weeks post-infection. Total cell counts suggested that there was no difference in the total number of cells in the gingiva, or the total number of CD19+ B cells in the gingiva from infected (PD) compared with sham-infected (CMC) mice (data not shown). The proportion of the cell population comprising B cells was similar in gingivae of control and infected mice (Fig. 1A, B). There was, however, an increase in the percentage of B cells expressing RANKL 1 week post-infection (5.36% CMC versus 11% mean PD, P < 0.01, Fig. 1A), suggesting that following P. gingivalis infection, the phenotype of B cells is altered. Further examination of the RANKLexpressing B cells was carried out based on expression of CD43, CD5 and CD23. This revealed that B1 B cells (CD19+, CD43+, CD5+ or CD5 ) cells expressed significantly more RANKL than B2 B cells (CD19+, CD43 , CD23+ or CD23 ) irrespective of infection with P. gingivalis (Fig. 1C). To determine whether the change in B-cell RANKL expression © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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Figure 1 Cells in the gingiva of mice following oral infection with Porphyromonas gingivalis. BALB/c mice were orally infected with P. gingivalis (PD, filled bars) or carrier alone (CMC, white bars). One (A) or six weeks (B and C) after infection, gingival tissues were harvested and cells were investigated by flow cytometry. The % of CD19-expressing cells within a ‘total cell gate’ based on forward and side scatter, was used to estimate the ‘total % B cells’ and within the B-cell population, the percentage of RANKL B cells was determined (A, B). RANKLexpressing B1 B cells (CD19+, CD43+, CD5+ or CD5 ) and B2 B cells (CD19+, CD43 , CD23+ or CD23 ) were similarly determined by flow cytometry (C). Data shown are mean standard deviation for three independent experiments, five mice pooled/group. **P < 0.01 by unpaired t-test; *P < 0.05 by analysis of variance with Tukey comparison.
and anti-P. gingivalis serum antibody (Fig. 3B) were significantly greater in the PD group compared with the CMC group. These changes in B-cell phenotype observed in this model of PD, suggest that B cells are activated and therefore could impact on the development of PD. To investigate whether overall, B cells have a pathological or a protective role in murine PD, B-cell-deficient lMT mice were orally infected with P. gingivalis and alveolar bone loss was assessed at 6 weeks post-infection (Fig. 4A). Infected lMT mice (lMT PD) had a similar level of alveolar bone to sham-infected lMT mice (lMT CMC). In contrast, infected wild-type mice (WT PD) demonstrated modest alveolar bone loss compared with sham-infected wild-type mice (WT control). This suggests that
observed in the gingiva reflected general B-cell activation, B-cell phenotype in the dLN was investigated. The proportions of germinal centre B cells, which are activated B cells that ultimately lead to antibody production, were evaluated by B-cell expression of Fas and GL7 (Dal Porto et al., 1998; Takahashi et al., 2001; Naito et al., 2007). B cells made up a greater proportion of the lymph node in infected mice compared with controls at both 1 and 6 weeks, which reached statistical significance at 6 weeks postinfection (Fig. 2). The percentage of germinal centre B cells was significantly increased at both 1 week (mean 2.03% CMC vs. mean 6.90% PD, P < 0.01) and 6 weeks (mean 4.45% CMC vs. mean 8.77% PD, P < 0.05) post-infection (Fig. 2). As anticipated, 6 weeks post-infection, alveolar bone loss (Fig. 3A)
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Figure 2 Cells in the cervical draining lymph nodes of mice following oral infection with Porphyromonas gingivalis. BALB/c mice were orally infected with P. gingivalis (PD, filled bars) or carrier alone (CMC, white bars). One (A) or six (B) weeks after infection, cervical draining lymph nodes were harvested and cells were investigated by flow cytometry. Data shown are mean standard deviation, five mice/group. *P < 0.05, **P < 0.01 relative to the CMC control group, by unpaired t-test.
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Figure 3 Alveolar bone loss and anti-Porphyromonas gingivalis antibody 6 weeks after oral infection with P. gingivalis. BALB/c mice were orally infected with P. gingivalis (PD, filled circles) or carrier alone (CMC, white circles). Six weeks after infection, jaws were defleshed and alveolar bone loss was assessed. Serum antibody was assessed by enzyme-linked immunosorbent assay. (A) Alveolar bone loss relative to CMC control, (B) serum IgG antibody specific for P. gingivalis. Each point represents data from a single animal, with four or five mice per group. Data shown are representative of two similar experiments. *P < 0.05, ***P < 0.01 relative to the control group, by unpaired t-test.
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Figure 4 Alveolar bone loss and anti-Porphyromonas gingivalis IgG antibody titers in wild-type and B-cell-deficient mice infected with P. gingivalis. Wild-type C57BL/6 mice (WT, circles) or lMT B-cell-deficient mice (uMT, squares) were infected with P. gingivalis W83 (PD, filled symbols), or carrier control (CMC, open symbols). Six weeks after infection alveolar bone loss and serum antibody were measured. (A) Alveolar bone level [bone loss estimated by measuring the distance from cemento–enamel junction (ACJ) to alveolar bone crest, normalized to WT CMC] and (B) serum anti-P. gingivalis IgG antibody titers in WT 6 weeks post-infection (data from one experiment, n = 5 per group for WT mice, n = 6 per group for lMT mice) relative to the WT CMC group mean. Data shown are mean per mouse (points) and mean for each group of mice (lines). ***P < 0.001 by one-way analysis of variance with a Tukey post hoc test, ns, not significant.
although lMT mice generally have less alveolar bone than WT mice, P. gingivalis infection did not induce further alveolar bone loss in the lMT mice. Anti-P. gingivalis serum IgG antibody titers in WT PD animals were significantly elevated compared with the WT CMC group (629.62 EU mean WT PD versus 1.73 EU mean WT CMC, P < 0.001). As expected, the lMT PD animals failed to raise an antibody response against P. gingivalis – with antibody titers detectable at very low background levels only, equivalent to the antibody detected in uninfected controls of either WT or lMT mice (Fig. 4B). To confirm that the inability of lMT mice to generate an anti-P. gingivalis antibody response was due to their inherent deficiency of B cells and plasma cells, lymphocyte composition in the dLN was assessed. As observed in normal BALB/c mice, the total number of B cells was significantly increased in 164
the dLN of the WT PD group relative to the WT CMC group (3,367,820 mean count CMC versus 8,320,240 mean count PD, P < 0.001). In comparison to the WT groups, the numbers of B cells present in the lMT groups were very low, irrespective of infection status. The absence of B cells had an impact on the T-cell response to infection in the lMT mice. WT PD mice had a significant increase in the percentage of CD4+ T cells that were CD44+ CD62L effector cells relative to the WT CMC group (10.01% mean CMC versus 12.46% mean PD, P < 0.001), but both the lMT CMC and the lMT PD mice had a proportion of T effector memory cells that was comparable with the WT CMC group (Fig. 5). Together, the data in Figures 4 and 5 indicate that B-cell-deficient mice have greater baseline alveolar bone loss, but an absence of P. gingivalis infectioninduced bone loss. This is associated with a lack of specific anti-P. gingivalis IgG antibody production, © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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which can be attributed to the deficiency of B cells in the dLN. DISCUSSION The oral microbiome and the host immune response are potentially equally culpable for the pathology in PD. Current PD treatment focuses on reducing the bacterial burden in PD patients. While undoubtedly successful in some patients, in many cases, this approach is associated with sub-optimal resolution and disease recurrence, hence a better understanding of the host response in PD may yield improvements in patient outcomes (Kinane et al., 2011). B cells are abundant in the inflammatory lesions in the gingiva of human PD patients and therefore could offer a therapeutic target (Berglundh & Donati, 2005). However, the overall impact of B cells on PD pathogenesis is not fully understood. Using a well-established murine model of PD we show that there are local and regional alterations in B cells following oral infection with P. gingivalis. There were apparently higher proportions of lymphocytes in the mouse oral mucosa than have been reported in human tissues, which may reflect our sample processing, which favored lymphocytes, or a true discrepancy between the species. There were no significant increases in cell numbers following infection, possibly a caveat of the model system. The murine gingival tissue samples encompass the diseased periodontal pocket but also include relatively extensive adjacent unaffected tissue compared A
with tissue samples taken from human periodontal pockets. Hence, potential differences between healthy and diseased sites in the mouse may be lost against the background of the rest of the tissue. Compared with findings in human tissue, we could only detect relatively low numbers of CD138 plasma cells in the murine gingivae (< 1% of total lymphocytes, data not shown), which is likely to reflect that the lesions studied in humans are of several years duration and this chronicity is not recaptured in this mouse model. As a result of limitations in cell number our studies are limited to cell phenotype when ideally cell function and phenotype would be characterized. Interestingly, when we looked at mature Bcell subsets in the gingiva (CD43+ B1, CD43 B2), we found that RANKL was differentially expressed among these, with the highest proportion of RANKLexpressing B cells in the B1 compartment, irrespective of infectious challenge. To the best of our knowledge, this is the first such characterization of gingival B cells in the mouse model. The significant upregulation of RANKL on gingival B cells is in broad agreement with previously published data. It has been demonstrated that blocking RANKL significantly reduces alveolar bone loss in other models of PD, and that transfer of activated RANKL-expressing B cells exacerbates alveolar bone loss in T-cell-deficient rodents infected with A. actinomycetemcomitans in a RANKL-dependent manner (Han et al., 2006, 2013; Yuan et al., 2011). The changes in B-cell populations observed in the draining lymph nodes are more pronounced than
B
Figure 5 Cell populations in the draining lymph node of lMT B-cell-deficient mice infected with Porphyromonas gingivalis. Wild-type C57BL/6 mice (WT, solid bars) or lMT B-cell-deficient (uMT, patterned bars) were infected with P. gingivalis W83 (PD, filled bars), or carrier control (CMC, open bars). Six weeks after infection cells in the draining lymph nodes were evaluated by flow cytometry. (A) The percentage of CD19+ B cells and (B) the percentage of CD4+ CD44+ CD62L effector memory T cells in the draining lymph node 6 weeks after infection (data from one experiment, n = 5 per group for WT mice, n = 6 per group for lMT mice). Data shown are mean standard deviation. *P < 0.05, ***P < 0.001 by one-way analysis of variance with a Tukey post hoc test.
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changes observed in the gingiva, indicating that following exposure to P. gingivalis, antigen reaches the lymph nodes draining the oral cavity in a form capable of inducing an immune response. There are early changes in B-cell activation evident within days of infection, but later only minimal changes in the local tissues, which may reflect the kinetics and lack of chronicity in the murine model. It remains to be elucidated whether lymphocytes migrate between the gingiva, the dLN and the circulation. The changes in the dLN are associated with generation of CD4+ effector memory T cells, and the production of systemically detectable class-switched antibody, commensurate with the concept of periodontal disease causing both local and systemic immune alteration with potential adverse consequences for general health (Moutsopoulos & Madianos, 2006). Periodontitis manifests from imbalance between protective immunity and harmful bystander damage to the periodontal structures. Gene knockout mice have proved useful for the dissection of immune responses in numerous infectious and inflammatory diseases. lMT mice, in which there is no membrane expression of IgM, show a defect in B-cell maturation caused by lack of signaling through the B-cell receptor. These mice have precursor cells or pre-B cells but lack mature B cells and plasma cells (Kitamura et al., 1991). We took advantage of these mice to determine if mature B cells are a critical mediator of bone loss associated with P. gingivalis infection. We found that lMT mice were protected from P. gingivalis infection induced alveolar bone loss (Fig. 3A). The lack of anti-P. gingivalis antibody in the lMT mice may be simply a result of lack of B cells or may imply a pathogenic role for antibody. Determining which aspects of B-cell function confer greater susceptibility to PD will require significant further investigation. In contrast to their response to infectious challenge, the lMT mice showed greater bone loss at baseline, suggesting that B cells may protect the alveolar bone in the steady state. This might be because natural low-affinity IgM and IgA antibodies are needed to prevent normally harmless commensal flora from causing tissue destruction. Alternatively, B cells may have roles in bone development, perhaps through their direct interaction with bone-forming cells (Brandtzaeg, 2013; Manilay & Zouali, 2014). For example, B cells in the bone marrow are 166
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responsible for 45% of the total production of osteoprotegerin (OPG) in the bone marrow (Li et al., 2007). The production of other bone morphogenic proteins by B cells outside the bone marrow and in pathological conditions has not, to our knowledge, been assessed in lMT mice. It is also conceivable that B-cell deficiency from birth may alter the nature of the microbiome at mucosal surfaces. These intriguing phenomena are under further investigation. Previously, Baker et al. (1994) attributed the reduction in alveolar bone loss observed in P. gingivalis-infected severe combined immunodeficient (SCID) mice (lacking both B cells and T cells) primarily to the deficiency of the CD4+ T-cell compartment, which they found to be responsible for the secretion of bone resorptive cytokines (Baker et al., 1999). While appreciating this role of CD4+ T cells in PD, we would argue, based on the data from our lMT experiment, that B cells also make an important contribution to P. gingivalisinduced bone loss. The contribution of B cells to P. gingivalis-induced bone loss was previously investigated in a murine model of incomplete B-cell deficiency – the IgD knockout (KO) mouse. It was demonstrated that IgD KO mice were also protected from P. gingivalis infection-induced alveolar bone loss. These mice still have mature B cells and can still make low levels of class-switched anti-P. gingivalis IgG, IgA and IgM. This, however, is delayed in response to infection. In this model, it was determined that the lack of T-cell activation in the IgD KO PD mice was accountable for the protection from alveolar bone loss. Parallels can be drawn with the lack of CD4 T-cell activation found in the IgD KO PD mice (Baker et al., 2009) and the lack of CD4+ effector T-cell generation observed in the dLN of our lMT PD mice. Although full analysis of the alterations in the T-cell compartment and their impact on disease is outwith the scope of the present study, in both IgD KO PD and the lMT cases this could be due to the requirement for B cells to present antigen and provide co-stimulation to cognate CD4+ T cells (Mollo et al., 2013). Although there is a reduction in T-cell activation, memory T cells can be detected in the lMT mice and presumably therefore these T cells have been activated by non-B-cell antigen-presenting cells. Different antigen-presenting cells, for example, dendritic cells, can support the © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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expansion of effector T cells but are not as effective as B cells. Other studies have demonstrated that expansion of T-cell clones is suboptimal in the absence of B cells (as observed here), and that this is specifically dependent on the expression of MHC II by B cells (Crawford et al., 2006). This is thought to be because B cells provide qualitatively different antigen presentation to dendritic cells in terms of provision of co-stimulation, signal strength, duration and cytokines, which results in B cells preferentially inducing a T helper type 2 phenotype in effector T cells (Macaulay et al., 1997). It has been postulated that B cells play an increasingly important role as antigenpresenting cells when the amount of antigen is limiting – as may be the case in our infection model. Interestingly, C57BL/6-Tcra mice which are T-cell deficient due to targeted deletion of the a chain of their T-cell receptors, were only partially protected from P. gingivalis infection-induced alveolar bone loss whereas the B-cell deficient IgD KO strain conferred total protection (Baker et al., 2002). Within the limitations of an in vivo model system, we have demonstrated that B cells make a substantial contribution to alveolar bone loss in murine PD. In mice infected with P. gingivalis we observed a significant increase in B-cell number and activation in the dLN, and also an increase in B-cell RANKL expression in the gingiva, observations that mirror reports of B-cell activation and RANKL expression in the inflamed gingiva of patients with PD. B-cell-deficient lMT mice were found to be protected from P. gingivalis infection-induced alveolar bone loss, although they demonstrated greater base-line alveolar bone loss. These studies provide a foundation for more mechanistic and detailed investigations to better understand the role of B cells in response to infection, and in physiological maintenance of mucosal surfaces and associated structures. This will be crucial to understanding the role played by immune activation in the oral cavity in terms of both oral and systemic disease, and to determine whether locally delivered, B-cell targeted therapies might be beneficial to PD patients. ACKNOWLEDGEMENTS This work was supported by ‘Gums&Joints’ EUFP7 Health agreement number 261460. AA is supported by Marie Curie ITN ‘RAPID’ 290246. LC is supported © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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by Sir Jules Thorn Studentship. SC supported by Scottish Funding Council Scottish Senior Clinical Fellowship Scheme. The authors acknowledge the assistance of the University of Glasgow Flow Cytometry Core Facility. CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES Apatzidou, D.A., Riggio, M.P. and Kinane, D.F. (2005) Impact of smoking on the clinical, microbiological and immunological parameters of adult patients with periodontitis. J Clin Periodontol 32: 973–983. Baker, P.J., Evans, R.T. and Roopenian, D.C. (1994) Oral infection with Porphyromonas gingivalis and induced alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Arch Oral Biol 39: 1035–1040. Baker, P.J., Dixon, M., Evans, R.T., Dufour, L., Johnson, E. and Roopenian, D.C. (1999) CD4+ T cells and the proinflammatory cytokines gamma interferon and interleukin-6 contribute to alveolar bone loss in mice. Infect Immun 67: 2804–2809. Baker, P.J., Howe, L., Garneau, J. and Roopenian, D.C. (2002) T cell knockout mice have diminished alveolar bone loss after oral infection with Porphyromonas gingivalis. FEMS Immunol Med Microbiol 34: 45–50. Baker, P.J., Boutaugh, N.R., Tiffany, M. and Roopenian, D.C. (2009) B Cell IgD deletion prevents alveolar bone loss following murine oral infection. Interdisc Persp Infect Dis 2009: 1–6. Belibasakis, G.N., Reddi, D. and Bostanci, N. (2011) Porphyromonas gingivalis induces RANKL in T-cells. Inflammation 34: 133–138. Berglundh, T. and Donati, M. (2005) Aspects of adaptive host response in periodontitis. J Clin Periodontol 32 (Suppl 6): 87–107. Berglundh, T., Liljenberg, B., Tarkowski, A. and Lindhe, J. (2002) The presence of local and circulating autoreactive B cells in patients with advanced periodontitis. J Clin Periodontol 29: 281–286. Brandtzaeg, P. (2013) Secretory immunity with special reference to the oral cavity. J Oral Microbiol 5: 20401. Cao, D., Khmaladze, I., JIA, H. et al. (2011) Pathogenic autoreactive B cells are not negatively selected toward matrix protein collagen II. J Immunol 187: 4451–4458.
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Kawai, T., Matsuyama, T., Hosokawa, Y. et al. (2006) B and T Lymphocytes Are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. Am J Pathol 169: 987–998. Kinane, D.F., Preshaw, P.M. and Loos, B.G. (2011) Hostresponse: understanding the cellular and molecular mechanisms of host-microbial interactions – Consensus of the Seventh European Workshop on Periodontology. J Clin Periodontol 38: 44–48. Kitamura, D., Roes, J., Kuhn, R. and Rajewsky, K. (1991) A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350: 423–426. Klareskog, L., Ronnelid, J., Lundberg, K., Padyukov, L. and Alfredsson, L. (2008) Immunity to citrullinated proteins in rheumatoid arthritis. Annu Rev Immunol 26: 651–675. Lappin, D.F., Apatzidou, D., Quirke, A.M. et al. (2013) Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anticitrullinated peptide antibody titres. J Clin Periodontol, 40: 907–915. Li, Y., Toraldo, G., Li, A. et al. (2007) B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo. Blood, 109: 3839–3848. Mackler, B.F., Frostad, K.B., Robertson, P.B. and Levy, B.M. (1977) Immunoglobulin bearing lymphocytes and plasma cells in human periodontal disease. J Periodontal Res, 12: 37–45. Macaulay, A.E., DeKruyff, R.H., Goodnow, C.C., Umetsu, D.T. (1997) Antigen-specific B cells preferentially induce CD4+ T cells to produce IL-4. J Immunol 158: 4171–4179. Manilay, J.O. and Zouali, M. (2014) Tight relationships between B lymphocytes and the skeletal system. Trends Mol Med 20: 405–412. Mollo, S.B., Zajac, A.J. and Harrington, L.E. (2013) Temporal requirements for B cells in the establishment of CD4 T cell memory. J Immunol 191: 6052–6059. Mooney, J., Adonogianaki, E. and Kinane, D.F. (1993) Relative avidity of serum antibodies to putative periodontopathogens in periodontal disease. J Periodontal Res 28: 444–450. Moutsopoulos, N.M. and Madianos, P.N. (2006) Lowgrade inflammation in chronic infectious diseases: paradigm of periodontal infections. Ann N Y Acad Sci 1088: 251–264. Naito, Y., Takematsu, H., Koyama, S. et al. (2007) Germinal center marker GL7 probes activation-dependent
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Periodontitis in the absence of B cells and specific anti-bacterial antibody J. Oliver-Bell1, J.P. Butcher2, J. Malcolm1, M.K.L. MacLeod1, A. Adrados Planell1, L. Campbell1, R.J.B. Nibbs1, P. Garside1, I.B. McInnes1 and S. Culshaw3 1 Institute of Infection, Immunology and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 2 Institute of Biomedical and Environmental Health Research, School of Science, University of the West of Scotland, Paisley, UK 3 Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
Correspondence: Shauna Culshaw, Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, UK Tel.: + 44 141 211 9733; E-mail: [email protected] Keywords: B cells; murine models; periodontitis Accepted 7 September 2014 DOI: 10.1111/omi.12082
SUMMARY Periodontitis (PD) results from complex interactions between a dysbiotic oral microbiota and a dysregulated host immune response. The inflammatory infiltrate in the gingiva of PD patients includes an abundance of B cells, implicating these cells in the immunopathology. We sought to investigate the role of B cells in PD using a murine model. Wild-type or B-cell-deficient (lMT) mice were orally infected with Porphyromonas gingivalis. One or six weeks following infection, lymphocyte populations in the gingiva and cervical draining lymph nodes (dLN) were analysed by flow cytometry; serum anti-P. gingivalis IgG antibody titers were measured by enzyme-linked immunosorbent assay, and alveolar bone loss was determined. In wild-type mice, the percentage of gingival B cells expressing receptor activator of nuclear factor-jB ligand (RANKL) was significantly increased 1 week post-infection (5.36% control versus 11% PD, P < 0.01). The percentage of Fas+ GL7+ germinal centre B cells in the dLN was significantly increased at both 1 week (2.03% control versus 6.90% PD, P < 0.01) and 6 weeks (4.45% control versus 8.77% PD, P < 0.05) postinfection. B-cell-deficient mice were protected from P. gingivalis-induced alveolar bone loss, 160
with a lack of B-cell proliferation and lack of CD4+ CD44+ CD62L T-cell generation in the dLN, and absence of serum anti-P. gingivalis antibodies. Our data imply a pathological role for B cells in PD, and that selective targeting of this immune axis may have a role in treating severe periodontal disease.
INTRODUCTION The inflammatory infiltrate in gingival tissue in periodontitis (PD) includes an abundance of B cells, along with their survival factors interleukin-6, A proliferation inducing ligand (APRIL) and B-cell activating factor (BAFF) (Gumus et al., 2014). Many studies have reported that plasma cells account for half of the leukocyte infiltrate, and B cells for a further 20% (Berglundh & Donati, 2005) In contrast, there are relatively few leukocytes in healthy gingivae, of which only about 5% are B cells (Gemmell et al., 2002). Tissue-resident plasma cells in patients with PD secrete antibodies of immunoglobulin G (IgG), IgA and IgM classes (Mackler et al., 1977; Seymour & Greenspan, 1979), including antibodies specific for bacteria associated with PD, including Porphyromon© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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as gingivalis (Apatzidou et al., 2005). Locally produced anti-P. gingivalis antibodies may contribute to the elevated titers detectable in the saliva and serum of patients. However, it is unclear whether these antiP. gingivalis antibodies are protective, destructive or irrelevant to disease status. Intriguingly, these antibodies appear more frequently in the serum of patients with concurrent rheumatoid arthritis, and may form part of the link between PD and rheumatoid arthritis (Kaur et al., 2013). Studies of PD patients before and after periodontal treatment, or studies comparing PD patients with healthy patients have consistently demonstrated a specific antibody response, but the role of that antibody remains difficult to define (Gmur et al., 1986; Takeuchi et al., 2006; Okada et al., 2013). The generally unresolving nature of chronic PD would imply that the on-going antibody production in a patient with disease is ineffective or insufficient in managing the oral microbiome. Moreover, the anti-bacterial humoral response is often accompanied by increased levels of local and circulating autoantibodies reactive to citrullinated peptides (Harvey et al., 2013; Lappin et al., 2013; de Pablo et al., 2014) and collagen (Hirsch et al., 1988). These autoantibodies may exacerbate tissue destruction and bone loss through the formation of immune complexes and the stimulation of Fc receptor bearing immune cells. The anti-citrulline autoantibodies found in patients with PD are hypothesized to offer some explanation of the link between PD and rheumatoid arthritis, the latter strongly associated with such antibodies (Klareskog et al., 2008). In addition to the generation of an inappropriate auto-antibody response, atypical activation of B cells could contribute to PD pathogenesis by their antigen-presentation, cytokine production, and expression and secretion of receptor activator of nuclear factor-jB ligand (RANKL), the latter promoting osteoclastogenesis via its receptor, RANK (Yasuda et al., 1999). RANKL is reported to be expressed predominantly by lymphocytes in the gingiva of PD patients (Kawai et al., 2006; Wara-Aswapati et al., 2007). A key role for RANKL has been documented in rodent models of PD (Yuan et al., 2011; Han et al., 2013), and in vitro studies indicate increased lymphocyte expression of RANKL following culture with Aggregatibacter actinomycetemcomitans or P. gingivalis (Han et al., 2009; Belibasakis et al., 2011). The requirement for antigen specificity in this system was demonstrated © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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in vivo by adoptive transfer of antigen-experienced B cells specific for A. actinomycetemcomitans, which exacerbated RANKL-dependent alveolar bone loss in rats immunized with the same bacteria (Han et al., 2006, 2009). In addition to RANKL expression, B cells have myriad other functions that may perpetuate disease. Compared with conventional ‘B2’ B cells, the ‘B1’ B cell subset possesses particular autoreactive and host-damaging potential. B1 cells are more likely to escape central tolerance, and are less dependent on help from T cells to become activated (Fagarasan et al., 2000; Cao et al., 2011). Small clinical studies have suggested recruitment or proliferation of B1 cells in the gingiva and circulation of PD patients (Berglundh et al., 2002; Donati et al., 2009). This role of B cells can be directly probed using murine models of B-cell abnormality and deficiency. We have investigated changes in the expression of RANKL and other cell surface markers by B cells in the gingiva and draining lymph node (dLN) of normal mice following infection with P. gingivalis, and investigated the role of mature B cells in PD using B-cell-deficient lMT mice. Our findings lend support to the hypothesis that B cells are activated, are a source of RANKL, and seem to play a predominantly pathological role in PD. METHODS Murine model of periodontitis Female BALB/c (Harlan, Bicester, UK), C57BL/6 (Charles River, Kent, UK), or lMT mice [bred in house on a C57BL/6 background; (Kitamura et al., 1991)] were maintained before and during experiments in specific pathogen-free conditions with ad libitum access to food and water at Biological Services, University of Glasgow. All work was performed under licence in accordance with UK Home Office regulations and after ethical approval from local research ethics committees. Porphyromonas gingivalis W83 (ATCC, Middlesex, UK) was prepared as previously described and mice were orally infected with 109 colony-forming units P. gingivalis W83 in 2% carboxymethylcellulose (CMC) by gavage on 3 or 4 consecutive days as previously described (Baker et al., 1994). Control mice received CMC alone. At 1 or 6 weeks post-infection, serum, maxillary teeth, 161
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gingiva and dLN were collected following terminal general anaesthesia and cardiac puncture.
Antibody titers were calculated as described previously (Gmur et al., 1986).
Flow cytometry
Assessment of alveolar bone loss
Gingival tissue was dissected from the maxillae – encompassing the palatal mucosa, and a very thin strip of buccal tissue obtained from the buccal surfaces. Tissue was collected into 1 ml phosphatebuffered saline, incubated with 500 lg Liberase DH (Roche, West Sussex, UK) at 37°C for 30 min, then 2 ml cRPMI (RPMI-1640 with 10% fetal calf serum, 1% penicillin streptomycin, 1% L-glutamine; Gibco, Invitrogen, Carlsbad, CA, USA) was added and tissue was dissociated using gentleMACS C tubes (Miltenyi Biotec, Surrey, UK) cellular dissociation protocol, resulting in between 2 9 105 and 5 9 105 viable cells per mouse assessed by trypan blue exclusion. In addition, cell suspensions were obtained from four cervical dLN from each mouse. For flow cytometry, cell suspensions were resuspended in Fc block (5% mouse serum in 2.4G2 hybridoma supernatant) then fluorochrome-labeled antibodies against CD45, CD95 (eBioscience, Hatfield, UK), CD19, CD43, CD23, CD5, RANKL, CD138, CD3, GL7 (BD Biosciences, San Jose, CA, USA) or similarly labeled isotype control antibodies. Cells were analysed using a MACS Quant (Miltenyi Biotec), and data were analysed using FLOWJO (Tree Star Inc., Ashland, OR, USA).
The maxilla was separated from the skull and gingivae were removed. Maxilla were defleshed and treated as previously described (Baker et al., 1994). Images were captured using an Olympus SZX7 stereo zoom microscope fitted with SC100 digital color camera. Measurements of the distance between the cemento–enamel junction and the alveolar bone crest were made using IMAGEJ software (National Institute of Health, Bethesda, MD, USA) to assess alveolar bone loss as previously described (Baker et al., 1994).
Enzyme-linked immunosorbent assays Interleukin-6 and tumour necrosis factor-a enzymelinked immunosorbent assays (ELISAs; ReadySET-Go! Kit, eBioscience, Hatfield, UK) and sRANKL ELISAs (DuoSet kit, R&D Systems, Abingdon, UK) were performed according to the manufacturer’s instructions. Antibody titers to P. gingivalis in the serum samples were ascertained as previously described (Mooney et al., 1993) with minor modification. In brief, Dynotech Immunolon 1B microtiter plates (ThermoFisher, Loughborough, UK) were coated overnight with heat-killed P. gingivalis W83 at an OD600 0.02 in 100 mM bicarbonate buffer pH 9.6. After blocking, serum dilutions in phosphate-buffered saline were added then bound antibody was detected with biotin-labeled anti-mouse-IgG antibodies, extravidin-horseradish peroxidase and 3,3’,5,5’-tetrameth ylbenzidine substrate (all from Sigma, Poole, UK). 162
Statistics Data were analysed by Student’s t-test or analysis of variance with Tukey comparison, as indicated in the figure legends, using GRAPHPAD PRISM 5 (La Jolla, CA, USA). RESULTS To evaluate changes in local B-cell populations during initial and later stages of the murine oral infection model of PD, cells were isolated from the gingiva of mice at 1 and 6 weeks post-infection. Total cell counts suggested that there was no difference in the total number of cells in the gingiva, or the total number of CD19+ B cells in the gingiva from infected (PD) compared with sham-infected (CMC) mice (data not shown). The proportion of the cell population comprising B cells was similar in gingivae of control and infected mice (Fig. 1A, B). There was, however, an increase in the percentage of B cells expressing RANKL 1 week post-infection (5.36% CMC versus 11% mean PD, P < 0.01, Fig. 1A), suggesting that following P. gingivalis infection, the phenotype of B cells is altered. Further examination of the RANKLexpressing B cells was carried out based on expression of CD43, CD5 and CD23. This revealed that B1 B cells (CD19+, CD43+, CD5+ or CD5 ) cells expressed significantly more RANKL than B2 B cells (CD19+, CD43 , CD23+ or CD23 ) irrespective of infection with P. gingivalis (Fig. 1C). To determine whether the change in B-cell RANKL expression © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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C
Figure 1 Cells in the gingiva of mice following oral infection with Porphyromonas gingivalis. BALB/c mice were orally infected with P. gingivalis (PD, filled bars) or carrier alone (CMC, white bars). One (A) or six weeks (B and C) after infection, gingival tissues were harvested and cells were investigated by flow cytometry. The % of CD19-expressing cells within a ‘total cell gate’ based on forward and side scatter, was used to estimate the ‘total % B cells’ and within the B-cell population, the percentage of RANKL B cells was determined (A, B). RANKLexpressing B1 B cells (CD19+, CD43+, CD5+ or CD5 ) and B2 B cells (CD19+, CD43 , CD23+ or CD23 ) were similarly determined by flow cytometry (C). Data shown are mean standard deviation for three independent experiments, five mice pooled/group. **P < 0.01 by unpaired t-test; *P < 0.05 by analysis of variance with Tukey comparison.
and anti-P. gingivalis serum antibody (Fig. 3B) were significantly greater in the PD group compared with the CMC group. These changes in B-cell phenotype observed in this model of PD, suggest that B cells are activated and therefore could impact on the development of PD. To investigate whether overall, B cells have a pathological or a protective role in murine PD, B-cell-deficient lMT mice were orally infected with P. gingivalis and alveolar bone loss was assessed at 6 weeks post-infection (Fig. 4A). Infected lMT mice (lMT PD) had a similar level of alveolar bone to sham-infected lMT mice (lMT CMC). In contrast, infected wild-type mice (WT PD) demonstrated modest alveolar bone loss compared with sham-infected wild-type mice (WT control). This suggests that
observed in the gingiva reflected general B-cell activation, B-cell phenotype in the dLN was investigated. The proportions of germinal centre B cells, which are activated B cells that ultimately lead to antibody production, were evaluated by B-cell expression of Fas and GL7 (Dal Porto et al., 1998; Takahashi et al., 2001; Naito et al., 2007). B cells made up a greater proportion of the lymph node in infected mice compared with controls at both 1 and 6 weeks, which reached statistical significance at 6 weeks postinfection (Fig. 2). The percentage of germinal centre B cells was significantly increased at both 1 week (mean 2.03% CMC vs. mean 6.90% PD, P < 0.01) and 6 weeks (mean 4.45% CMC vs. mean 8.77% PD, P < 0.05) post-infection (Fig. 2). As anticipated, 6 weeks post-infection, alveolar bone loss (Fig. 3A)
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Figure 2 Cells in the cervical draining lymph nodes of mice following oral infection with Porphyromonas gingivalis. BALB/c mice were orally infected with P. gingivalis (PD, filled bars) or carrier alone (CMC, white bars). One (A) or six (B) weeks after infection, cervical draining lymph nodes were harvested and cells were investigated by flow cytometry. Data shown are mean standard deviation, five mice/group. *P < 0.05, **P < 0.01 relative to the CMC control group, by unpaired t-test.
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Figure 3 Alveolar bone loss and anti-Porphyromonas gingivalis antibody 6 weeks after oral infection with P. gingivalis. BALB/c mice were orally infected with P. gingivalis (PD, filled circles) or carrier alone (CMC, white circles). Six weeks after infection, jaws were defleshed and alveolar bone loss was assessed. Serum antibody was assessed by enzyme-linked immunosorbent assay. (A) Alveolar bone loss relative to CMC control, (B) serum IgG antibody specific for P. gingivalis. Each point represents data from a single animal, with four or five mice per group. Data shown are representative of two similar experiments. *P < 0.05, ***P < 0.01 relative to the control group, by unpaired t-test.
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Figure 4 Alveolar bone loss and anti-Porphyromonas gingivalis IgG antibody titers in wild-type and B-cell-deficient mice infected with P. gingivalis. Wild-type C57BL/6 mice (WT, circles) or lMT B-cell-deficient mice (uMT, squares) were infected with P. gingivalis W83 (PD, filled symbols), or carrier control (CMC, open symbols). Six weeks after infection alveolar bone loss and serum antibody were measured. (A) Alveolar bone level [bone loss estimated by measuring the distance from cemento–enamel junction (ACJ) to alveolar bone crest, normalized to WT CMC] and (B) serum anti-P. gingivalis IgG antibody titers in WT 6 weeks post-infection (data from one experiment, n = 5 per group for WT mice, n = 6 per group for lMT mice) relative to the WT CMC group mean. Data shown are mean per mouse (points) and mean for each group of mice (lines). ***P < 0.001 by one-way analysis of variance with a Tukey post hoc test, ns, not significant.
although lMT mice generally have less alveolar bone than WT mice, P. gingivalis infection did not induce further alveolar bone loss in the lMT mice. Anti-P. gingivalis serum IgG antibody titers in WT PD animals were significantly elevated compared with the WT CMC group (629.62 EU mean WT PD versus 1.73 EU mean WT CMC, P < 0.001). As expected, the lMT PD animals failed to raise an antibody response against P. gingivalis – with antibody titers detectable at very low background levels only, equivalent to the antibody detected in uninfected controls of either WT or lMT mice (Fig. 4B). To confirm that the inability of lMT mice to generate an anti-P. gingivalis antibody response was due to their inherent deficiency of B cells and plasma cells, lymphocyte composition in the dLN was assessed. As observed in normal BALB/c mice, the total number of B cells was significantly increased in 164
the dLN of the WT PD group relative to the WT CMC group (3,367,820 mean count CMC versus 8,320,240 mean count PD, P < 0.001). In comparison to the WT groups, the numbers of B cells present in the lMT groups were very low, irrespective of infection status. The absence of B cells had an impact on the T-cell response to infection in the lMT mice. WT PD mice had a significant increase in the percentage of CD4+ T cells that were CD44+ CD62L effector cells relative to the WT CMC group (10.01% mean CMC versus 12.46% mean PD, P < 0.001), but both the lMT CMC and the lMT PD mice had a proportion of T effector memory cells that was comparable with the WT CMC group (Fig. 5). Together, the data in Figures 4 and 5 indicate that B-cell-deficient mice have greater baseline alveolar bone loss, but an absence of P. gingivalis infectioninduced bone loss. This is associated with a lack of specific anti-P. gingivalis IgG antibody production, © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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which can be attributed to the deficiency of B cells in the dLN. DISCUSSION The oral microbiome and the host immune response are potentially equally culpable for the pathology in PD. Current PD treatment focuses on reducing the bacterial burden in PD patients. While undoubtedly successful in some patients, in many cases, this approach is associated with sub-optimal resolution and disease recurrence, hence a better understanding of the host response in PD may yield improvements in patient outcomes (Kinane et al., 2011). B cells are abundant in the inflammatory lesions in the gingiva of human PD patients and therefore could offer a therapeutic target (Berglundh & Donati, 2005). However, the overall impact of B cells on PD pathogenesis is not fully understood. Using a well-established murine model of PD we show that there are local and regional alterations in B cells following oral infection with P. gingivalis. There were apparently higher proportions of lymphocytes in the mouse oral mucosa than have been reported in human tissues, which may reflect our sample processing, which favored lymphocytes, or a true discrepancy between the species. There were no significant increases in cell numbers following infection, possibly a caveat of the model system. The murine gingival tissue samples encompass the diseased periodontal pocket but also include relatively extensive adjacent unaffected tissue compared A
with tissue samples taken from human periodontal pockets. Hence, potential differences between healthy and diseased sites in the mouse may be lost against the background of the rest of the tissue. Compared with findings in human tissue, we could only detect relatively low numbers of CD138 plasma cells in the murine gingivae (< 1% of total lymphocytes, data not shown), which is likely to reflect that the lesions studied in humans are of several years duration and this chronicity is not recaptured in this mouse model. As a result of limitations in cell number our studies are limited to cell phenotype when ideally cell function and phenotype would be characterized. Interestingly, when we looked at mature Bcell subsets in the gingiva (CD43+ B1, CD43 B2), we found that RANKL was differentially expressed among these, with the highest proportion of RANKLexpressing B cells in the B1 compartment, irrespective of infectious challenge. To the best of our knowledge, this is the first such characterization of gingival B cells in the mouse model. The significant upregulation of RANKL on gingival B cells is in broad agreement with previously published data. It has been demonstrated that blocking RANKL significantly reduces alveolar bone loss in other models of PD, and that transfer of activated RANKL-expressing B cells exacerbates alveolar bone loss in T-cell-deficient rodents infected with A. actinomycetemcomitans in a RANKL-dependent manner (Han et al., 2006, 2013; Yuan et al., 2011). The changes in B-cell populations observed in the draining lymph nodes are more pronounced than
B
Figure 5 Cell populations in the draining lymph node of lMT B-cell-deficient mice infected with Porphyromonas gingivalis. Wild-type C57BL/6 mice (WT, solid bars) or lMT B-cell-deficient (uMT, patterned bars) were infected with P. gingivalis W83 (PD, filled bars), or carrier control (CMC, open bars). Six weeks after infection cells in the draining lymph nodes were evaluated by flow cytometry. (A) The percentage of CD19+ B cells and (B) the percentage of CD4+ CD44+ CD62L effector memory T cells in the draining lymph node 6 weeks after infection (data from one experiment, n = 5 per group for WT mice, n = 6 per group for lMT mice). Data shown are mean standard deviation. *P < 0.05, ***P < 0.001 by one-way analysis of variance with a Tukey post hoc test.
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changes observed in the gingiva, indicating that following exposure to P. gingivalis, antigen reaches the lymph nodes draining the oral cavity in a form capable of inducing an immune response. There are early changes in B-cell activation evident within days of infection, but later only minimal changes in the local tissues, which may reflect the kinetics and lack of chronicity in the murine model. It remains to be elucidated whether lymphocytes migrate between the gingiva, the dLN and the circulation. The changes in the dLN are associated with generation of CD4+ effector memory T cells, and the production of systemically detectable class-switched antibody, commensurate with the concept of periodontal disease causing both local and systemic immune alteration with potential adverse consequences for general health (Moutsopoulos & Madianos, 2006). Periodontitis manifests from imbalance between protective immunity and harmful bystander damage to the periodontal structures. Gene knockout mice have proved useful for the dissection of immune responses in numerous infectious and inflammatory diseases. lMT mice, in which there is no membrane expression of IgM, show a defect in B-cell maturation caused by lack of signaling through the B-cell receptor. These mice have precursor cells or pre-B cells but lack mature B cells and plasma cells (Kitamura et al., 1991). We took advantage of these mice to determine if mature B cells are a critical mediator of bone loss associated with P. gingivalis infection. We found that lMT mice were protected from P. gingivalis infection induced alveolar bone loss (Fig. 3A). The lack of anti-P. gingivalis antibody in the lMT mice may be simply a result of lack of B cells or may imply a pathogenic role for antibody. Determining which aspects of B-cell function confer greater susceptibility to PD will require significant further investigation. In contrast to their response to infectious challenge, the lMT mice showed greater bone loss at baseline, suggesting that B cells may protect the alveolar bone in the steady state. This might be because natural low-affinity IgM and IgA antibodies are needed to prevent normally harmless commensal flora from causing tissue destruction. Alternatively, B cells may have roles in bone development, perhaps through their direct interaction with bone-forming cells (Brandtzaeg, 2013; Manilay & Zouali, 2014). For example, B cells in the bone marrow are 166
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responsible for 45% of the total production of osteoprotegerin (OPG) in the bone marrow (Li et al., 2007). The production of other bone morphogenic proteins by B cells outside the bone marrow and in pathological conditions has not, to our knowledge, been assessed in lMT mice. It is also conceivable that B-cell deficiency from birth may alter the nature of the microbiome at mucosal surfaces. These intriguing phenomena are under further investigation. Previously, Baker et al. (1994) attributed the reduction in alveolar bone loss observed in P. gingivalis-infected severe combined immunodeficient (SCID) mice (lacking both B cells and T cells) primarily to the deficiency of the CD4+ T-cell compartment, which they found to be responsible for the secretion of bone resorptive cytokines (Baker et al., 1999). While appreciating this role of CD4+ T cells in PD, we would argue, based on the data from our lMT experiment, that B cells also make an important contribution to P. gingivalisinduced bone loss. The contribution of B cells to P. gingivalis-induced bone loss was previously investigated in a murine model of incomplete B-cell deficiency – the IgD knockout (KO) mouse. It was demonstrated that IgD KO mice were also protected from P. gingivalis infection-induced alveolar bone loss. These mice still have mature B cells and can still make low levels of class-switched anti-P. gingivalis IgG, IgA and IgM. This, however, is delayed in response to infection. In this model, it was determined that the lack of T-cell activation in the IgD KO PD mice was accountable for the protection from alveolar bone loss. Parallels can be drawn with the lack of CD4 T-cell activation found in the IgD KO PD mice (Baker et al., 2009) and the lack of CD4+ effector T-cell generation observed in the dLN of our lMT PD mice. Although full analysis of the alterations in the T-cell compartment and their impact on disease is outwith the scope of the present study, in both IgD KO PD and the lMT cases this could be due to the requirement for B cells to present antigen and provide co-stimulation to cognate CD4+ T cells (Mollo et al., 2013). Although there is a reduction in T-cell activation, memory T cells can be detected in the lMT mice and presumably therefore these T cells have been activated by non-B-cell antigen-presenting cells. Different antigen-presenting cells, for example, dendritic cells, can support the © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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expansion of effector T cells but are not as effective as B cells. Other studies have demonstrated that expansion of T-cell clones is suboptimal in the absence of B cells (as observed here), and that this is specifically dependent on the expression of MHC II by B cells (Crawford et al., 2006). This is thought to be because B cells provide qualitatively different antigen presentation to dendritic cells in terms of provision of co-stimulation, signal strength, duration and cytokines, which results in B cells preferentially inducing a T helper type 2 phenotype in effector T cells (Macaulay et al., 1997). It has been postulated that B cells play an increasingly important role as antigenpresenting cells when the amount of antigen is limiting – as may be the case in our infection model. Interestingly, C57BL/6-Tcra mice which are T-cell deficient due to targeted deletion of the a chain of their T-cell receptors, were only partially protected from P. gingivalis infection-induced alveolar bone loss whereas the B-cell deficient IgD KO strain conferred total protection (Baker et al., 2002). Within the limitations of an in vivo model system, we have demonstrated that B cells make a substantial contribution to alveolar bone loss in murine PD. In mice infected with P. gingivalis we observed a significant increase in B-cell number and activation in the dLN, and also an increase in B-cell RANKL expression in the gingiva, observations that mirror reports of B-cell activation and RANKL expression in the inflamed gingiva of patients with PD. B-cell-deficient lMT mice were found to be protected from P. gingivalis infection-induced alveolar bone loss, although they demonstrated greater base-line alveolar bone loss. These studies provide a foundation for more mechanistic and detailed investigations to better understand the role of B cells in response to infection, and in physiological maintenance of mucosal surfaces and associated structures. This will be crucial to understanding the role played by immune activation in the oral cavity in terms of both oral and systemic disease, and to determine whether locally delivered, B-cell targeted therapies might be beneficial to PD patients. ACKNOWLEDGEMENTS This work was supported by ‘Gums&Joints’ EUFP7 Health agreement number 261460. AA is supported by Marie Curie ITN ‘RAPID’ 290246. LC is supported © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 160–169
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by Sir Jules Thorn Studentship. SC supported by Scottish Funding Council Scottish Senior Clinical Fellowship Scheme. The authors acknowledge the assistance of the University of Glasgow Flow Cytometry Core Facility. CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES Apatzidou, D.A., Riggio, M.P. and Kinane, D.F. (2005) Impact of smoking on the clinical, microbiological and immunological parameters of adult patients with periodontitis. J Clin Periodontol 32: 973–983. Baker, P.J., Evans, R.T. and Roopenian, D.C. (1994) Oral infection with Porphyromonas gingivalis and induced alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Arch Oral Biol 39: 1035–1040. Baker, P.J., Dixon, M., Evans, R.T., Dufour, L., Johnson, E. and Roopenian, D.C. (1999) CD4+ T cells and the proinflammatory cytokines gamma interferon and interleukin-6 contribute to alveolar bone loss in mice. Infect Immun 67: 2804–2809. Baker, P.J., Howe, L., Garneau, J. and Roopenian, D.C. (2002) T cell knockout mice have diminished alveolar bone loss after oral infection with Porphyromonas gingivalis. FEMS Immunol Med Microbiol 34: 45–50. Baker, P.J., Boutaugh, N.R., Tiffany, M. and Roopenian, D.C. (2009) B Cell IgD deletion prevents alveolar bone loss following murine oral infection. Interdisc Persp Infect Dis 2009: 1–6. Belibasakis, G.N., Reddi, D. and Bostanci, N. (2011) Porphyromonas gingivalis induces RANKL in T-cells. Inflammation 34: 133–138. Berglundh, T. and Donati, M. (2005) Aspects of adaptive host response in periodontitis. J Clin Periodontol 32 (Suppl 6): 87–107. Berglundh, T., Liljenberg, B., Tarkowski, A. and Lindhe, J. (2002) The presence of local and circulating autoreactive B cells in patients with advanced periodontitis. J Clin Periodontol 29: 281–286. Brandtzaeg, P. (2013) Secretory immunity with special reference to the oral cavity. J Oral Microbiol 5: 20401. Cao, D., Khmaladze, I., JIA, H. et al. (2011) Pathogenic autoreactive B cells are not negatively selected toward matrix protein collagen II. J Immunol 187: 4451–4458.
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