Periodontitis Bone Loss in Experimental Murine Dependent

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The B Cell−Stimulatory Cytokines BLyS and APRIL Are Elevated in Human Periodontitis and Are Required for B Cell−Dependent Bone Loss in Experimental Murine This information is current as Periodontitis of September 28, 2021. Toshiharu Abe, Mohammed AlSarhan, Manjunatha R. Benakanakere, Tomoki Maekawa, Denis F. Kinane, Michael P. Cancro, Jonathan M. Korostoff and George Hajishengallis J Immunol 2015; 195:1427-1435; Prepublished online 6 July Downloaded from 2015; doi: 10.4049/jimmunol.1500496 http://www.jimmunol.org/content/195/4/1427 http://www.jimmunol.org/

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The B Cell–Stimulatory Cytokines BLyS and APRIL Are Elevated in Human Periodontitis and Are Required for B Cell–Dependent Bone Loss in Experimental Murine Periodontitis

Toshiharu Abe,*,1 Mohammed AlSarhan,†,1,2 Manjunatha R. Benakanakere,† Tomoki Maekawa,* Denis F. Kinane,† Michael P. Cancro,‡ Jonathan M. Korostoff,† and George Hajishengallis*

B-lineage cells (B lymphocytes and plasma cells) predominate in the inflammatory infiltrate of human chronic periodontitis. How- ever, their role in disease pathogenesis and the factors responsible for their persistence in chronic lesions are poorly understood. In Downloaded from this regard, two cytokines of the TNF ligand superfamily, a proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), are important for the survival, proliferation, and maturation of B cells. Thus, we hypothesized that APRIL and/or BLyS are upregulated in periodontitis and contribute to induction of periodontal bone loss. This hypothesis was addressed in both human and mouse experimental systems. We show that, relative to healthy controls, the expression of APRIL and BLyS mRNA and protein was upregulated in natural and experimental periodontitis in humans and mice, respectively. The elevated expression of these cytokines correlated with increased numbers of B cells/plasma cells in both species. Moreover, APRIL and BLyS partially colocalized with k L http://www.jimmunol.org/ chain-expressing B-lineage cells at the epithelial–connective tissue interface. Ligature-induced periodontitis resulted in significantly less bone loss in B cell–deficient mice compared with wild-type controls. Ab-mediated neutralization of APRIL or BLyS diminished the number of B cells in the gingival tissue and inhibited bone loss in wild-type, but not in B cell-deficient, mice. In conclusion, B cells and specific cytokines involved in their growth and differentiation contribute to periodontal bone loss. Moreover, APRIL and BLyS have been identified as potential therapeutic targets in periodontitis. The Journal of Immunology, 2015, 195: 1427–1435.

eriodontitis is a prevalent chronic inflammatory disease typ- sufficient to cause disease; it is actually the host inflammatory re- by guest on September 28, 2021 ified by destruction of the tooth-supporting tissues (gingiva, sponse to this polymicrobial challenge that ultimately induces tissue P periodontal ligament, and alveolar bone) and is associated damage and bone loss, the hallmark of periodontitis (2). A number of with increased risk for certain systemic disorders (e.g., atherosclerosis inflammatory and stromal cell types have been implicated in this and rheumatoid arthritis) (1). Although the pathogenesis of peri- destructive process (3–6). However, the role of plasma cells and their odontitis involves polymicrobial synergy and dysbiosis, the bacterial precursors, B lymphocytes, remains quite enigmatic, despite their communities of the tooth-associated biofilm (dental plaque) are not high abundance in advanced periodontal lesions (7–9). Indeed, plasma and B cells together make up ∼60% of the total leukocytes *Department of Microbiology, Penn Dental Medicine, University of Pennsylvania, present in periodontal lesions associated with bone loss (8). Philadelphia, PA 19104; †Department of Periodontics, Penn Dental Medicine, Uni- Through their role in humoral immunity, B cells/plasma cells versity of Pennsylvania, Philadelphia, PA 19104; and ‡Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, could, in principle, have a protective function in periodontitis. For Philadelphia, PA 19104 instance, the Ab response could contribute to the control of the 1T.A. and M.A. contributed equally to this work. dysbiotic microbiota in periodontal pockets and prevent bacterial 2Current address: Department of Periodontics and Community Dentistry, College of invasion into the gingival connective tissue, thereby limiting Dentistry at King Saud University, Riyadh, Saudi Arabia. inflammation and disease. However, it is well established that ORCID: 0000-0001-7392-8852 (G.H.). periodontitis progresses, despite the induction of specific Ab Received for publication March 2, 2015. Accepted for publication June 12, 2015. responses to periodontal bacteria in affected individuals. This is This work was supported by National Institutes of Health Grants DE015254, likely due to the low affinity of the Abs and/or their unfavorable DE017138, DE021685, DE024716, AI068730 (all to G.H.), and DE023836 (to functional characteristics (e.g., poor opsonophagocytic capacity) D.F.K.). M.A. was supported by funds (Grant 4/52/146299) from the College of Dentistry Research Center and Deanship of Scientific Research at King Saud University. (5, 10, 11). In fact, it is thought that the B cell–mediated immune response, and specifically an IgG-dominant response, might Address correspondence and reprint requests to Dr. George Hajishengallis or Dr. Jonathan M. Korostoff, School of Dental Medicine, University of Pennsylvania, contribute to the pathogenesis of periodontitis. In this regard, 240 South 40th Street, Philadelphia, PA 19104-6030 (G.H.) or Department of Peri- advanced periodontitis is associated with increased deposition odontics, Penn Dental Medicine, University of Pennsylvania, 240 South 40th Street, Philadelphia, PA 19104 (J.M.K.). E-mail addresses: [email protected] (G.H.) or of IgG immune complexes along with complement activation [email protected] (J.M.K.) fragments (12, 13). Most importantly, B cells constitute a major Abbreviations used in this article: ABC, alveolar bone crest; APRIL, a proliferation- source of membrane-bound and secreted receptor activator of inducing ligand; BLyS, B-lymphocyte stimulator; CEJ, cementoenamel junction; KO, NF-kB ligand (RANKL) in the bone resorptive lesions of hu- knockout; RANKL, receptor activator of NF-kB ligand; WT, wild-type. man periodontitis (14). In this regard, it was shown that .90% Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 of B cells in periodontitis lesions express RANKL, whereas the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500496 1428 BLyS AND APRIL IN ALVEOLAR BONE LOSS percentage of RANKL+ B cells in healthy gingiva is negligible Materials and Methods (14). Given that RANKL plays a key role in osteoclast differ- Human subjects and gingival tissue specimens entiation and activation (15), it is plausible that B cells partici- Research was performed under an Institutional Review Board–approved pate in the induction of pathological bone loss in periodontitis. protocol. Signed informed consent was obtained to procure normally dis- The notion that B cells mediate destructive effects in peri- carded gingival tissue from individuals treated in the postgraduate clinic of odontitis is consistent with findings from previous studies in the Department of Periodontics, University of Pennsylvania School of Dental animal models. Adoptive transfer of bacterial Ag-specific B cells Medicine. All subjects were 18 y of age or older with no serious medical into rats, followed by local oral exposure to the same bacterium illness contraindicative of surgical treatment. Diseased tissue was obtained from 14 patients undergoing pocket-reduction surgery to treat chronic peri- (Aggregatibacter actinomycetemcomitans), leads to RANKL- odontitis. Control tissue specimens were obtained from 14 clinically healthy dependent periodontal bone loss (16, 17). Despite the artificial subjects who required crown-lengthening surgery. The mean demographic priming approach involved, these studies indicated that specific and clinical parameters of the two groups are shown in Table I. The inclusion Ag-triggered B cells can potentially provoke bone resorption. criteria for chronic periodontitis were diseased sites with probing pocket depth $ 5 mm associated with clinical attachment loss $ 4 mm, presence of The role of B cells in periodontitis was addressed more recently bleeding on probing, and radiographic evidence of bone loss. Sites with in studies using a model of the disease induced by oral gavage probing pocket depth # 3 mm, no clinical attachment loss, absence of with Porphyromonas gingivalis in wild-type (WT) and B cell– bleeding on probing, and normal radiographic bone levels were used as knockout (KO) mice. An investigation by Zhu et al. (18) did not healthy controls. All of the periodontitis patients underwent scaling and root support a role for naturally occurring B cells in periodontal planing 1–2 mo prior to the day of surgery. disease pathogenesis, unless the mice were fed a high-fat diet. Mice

However, this study did not detect B cells in the periodontal tm1Cgn Downloaded from B6.129S2-Ighm /J mice constitute a model of B cell immunodeficiency tissue of normal (nonobese) mice (18), suggesting that the model and were purchased from The Jackson Laboratory, along with C57BL/6J WT used or the specific experimental conditions do not readily controls. B6.129S2-Ighmtm1Cgn/J mice, referred to in this article as B cell– support the development of a B cell infiltrate that is character- KO mice, do not express membrane-bound IgM; consequently, B cell de- istic of human periodontitis. In contrast, Oliver-Bell et al. (19) velopment is blocked at the pro-B stage (28). WT and B cell–KO mice were housed in a pathogen-free environment and used when they were 10–12 wk showed that B cell–KO mice are protected from P. gingivalis– old. All animal procedures were performed according to protocols approved induced bone loss relative to WT controls. In sum, correlative by the Institutional Animal Care and Use Committee of the University of http://www.jimmunol.org/ human studies and certain animal model–based studies provide Pennsylvania. suggestive evidence for B cell involvement in plaque-induced Quantitative real-time PCR periodontal bone loss. Two cytokines of the TNF superfamily, B-lymphocyte stim- Total RNA was extracted from human or mouse gingival tissue using the MELT ulator (BLyS; also known as B cell–activating factor, TNFSF13B, Total Nucleic Acid Isolation System (Life Technologies) or RNeasy Mini Kit (QIAGEN) and quantified by spectrometry at 260 and 280 nm. The RNA was or TALL-1) and a proliferation-inducing ligand (APRIL; also reverse transcribed using the High-Capacity RNA-to-cDNA kit (Life Tech- known as TNFSF13 or TALL-2) are important contributors to the nologies), and real-time PCR with cDNA was performed using the ABI 7500 survival and maturation of B cells (20, 21). APRIL and BLyS are Fast System, according to the manufacturer’s protocol (Life Technologies). type II transmembrane proteins that are proteolytically cleaved, TaqMan probes, sense primers, and antisense primers for detection and by guest on September 28, 2021 either intracellularly (APRIL) or at the cell membrane (BLyS), to quantification of GAPDH (normalization control) and cytokine genes investi- gatedinthisstudy(seeFigs.1,7A)were purchased from Life Technologies. generate active secreted forms (21, 22). Upon secretion, APRIL Data were analyzed using the comparative (DDCt) method. and BLyS can interact with two common receptors found on B cells and plasma cells, the B cell–maturation Ag and trans- Immunofluorescence histochemistry membrane activator and cyclophilin ligand interactor (21, 23). Human tissues. Serial sections (8–10 mm thick) of formaldehyde-fixed In addition, BLyS interacts with a third receptor, the so-called OCT-embedded gingival tissue were stained with H&E to examine the his- tological features under light microscopy or were processed for immunohis- “B cell-activating factor receptor” (21, 23). The B cell–matu- tochemistry, as previously described (29), to detect the expression of APRIL, ration Ag promotes the survival of plasma cells, transmembrane BLyS, and Ig k L chain. To this end, the following Abs were used: goat anti- activator and cyclophilin ligand interactor supports T cell–in- human APRIL polyclonal IgG (Abcam), goat anti-human BLyS polyclonal dependent B cell Ab responses and isotype switching, and B cell– IgG (R&D Systems), and FITC-conjugated rabbit anti-human Ig k Lchain activating factor receptor is critical for survival and maturation of polyclonal IgG (DakoCytomation) that recognizes B cells and Ig-secreting plasma cells (30). Alexa Fluor 594–conjugated donkey anti-goat IgG was used immature B cells. Accordingly, APRIL-deficient mice exhibit im- as a secondary reagent for the unconjugated primary Abs. Human tonsil paired mucosal humoral immunity (24), and BLyS-deficient mice specimens, purchased from Newcomer Supply, were used as positive control display impaired B cell maturation (25). for Ig k L chain staining. Slides were mounted with cover slips using ProLong Although APRIL and BLyS have been implicated in autoim- Gold Antifade reagent containing DAPI (Life Technologies). munity and neoplastic B cell transformation (23, 26), their role Mouse tissues. Maxillae with intact surrounding tissue were fixed in 4% paraformaldehyde, decalcified in Immunocal solution (Decal Chemical) for in periodontal disease pathogenesis has not been addressed. We 14 d, placed in Cal-Arrest solution (Decal Chemical) to neutralize the pH of reasoned that APRIL and/or BLyS is required for the mainte- the tissue, and embedded in OCT compound. Coronal sections (6-mmthick) nance of the large number of B cells and plasma cells typically were stained with mAbs to CD19 (6D5), CD138 (EPR6454), or BLyS (Buffy found in periodontitis lesions. Therefore, we hypothesized that 2) or with polyclonal Ab to APRIL (all from Abcam), followed by appropriate these B cell–stimulatory cytokines are upregulated in periodon- secondary reagents conjugated to Alexa Fluor 594 or Alexa Fluor 488 (Life Technologies). Staining for Igs in tissues was performed using Alexa Fluor titis and play a role in disease pathogenesis. This hypothesis 488–conjugated goat anti-mouse IgM (m chain) and Alexa Fluor 594–conju- was addressed in both human and mouse experimental systems; gated goat anti-mouse IgG (H+L) (Life Technologies). the former allowed us to obtain clinically relevant information, The specificity of staining was confirmed using appropriate isotype- and the latter enabled us to perform cause-and-effect experi- control or nonimmune IgG. Stained images were visualized and captured using a Nikon Eclipse Ni-E automated fluorescent microscope and NIS- ments that cannot be typically addressed in human studies (27). Elements software. Our study confirmed the hypothesis and showed that, in the presence of APRIL and BLyS, B cells are causally linked to Ligature-induced periodontitis and intervention experiments periodontal bone loss, thereby identifying potential B cell–targeted The placement of ligatures in conventional (but not germ-free) mice accel- treatments for periodontitis. erates bacteria-mediated inflammation and bone loss (31, 32). To induce bone The Journal of Immunology 1429 loss, a 5-0 silk ligature was tied around the maxillary left second molar, as previously described (33). The contralateral molar tooth in each mouse was left unligated to serve as a baseline control for bone loss measurements. The ligatures remained in place in all mice throughout the experimental period. The mice were euthanized at various time points (0–10 d) after placement of the ligatures, and defleshed maxillae were used to measure bone heights (i.e., the distances from the cementoenamel junction [CEJ] to the alveolar bone crest [ABC]) under a Nikon SMZ800 microscope using a 403 objective. Images of the maxillae were captured using a Nikon Digital Sight DS-U3 camera con- troller, and CEJ–ABC distances were measured at six predetermined points on the ligated molar and adjacent regions using NIS-Elements software (33). To calculate bone loss, the six-site total CEJ–ABC distance for the ligated side of each mouse was subtracted from the six-site total CEJ–ABC distance of the contralateral unligated side. Negative values indicated bone loss relative to the baseline (unligated control). In intervention experiments, blocking mAbs to FIGURE 1. Upregulation of APRIL and BLyS mRNA expression in APRIL (Apry-1-1; IgG2b) or BLyS (Sandy-2; IgG1; both from Adipogen) or gingival biopsy specimens from periodontitis patients relative to healthy their isotype controls were microinjected into the palatal gingiva of the ligated controls. Gingival mRNA expression of APRIL, BLyS, RANKL, and second maxillary molar, as previously described (34, 35). Each animal received osteoprotegerin (OPG) (the latter two molecules were included for com- 5 mg mAb or isotype control through microinjections performed on day 4 parative reasons) in healthy and diseased sites was determined by quan- following placement of ligatures and once daily thereafter, until the day before sacrifice (performed on day 10). titative real-time PCR. Results were normalized against GAPDH mRNA and are presented as fold change in the transcript levels in diseased sites

Statistical analysis relative to those of healthy sites, which were assigned an average value of Downloaded from 1. Dots represent individual values (n = 14 subjects/group), and each box Data were evaluated by ANOVA and the Dunnett multiple comparison test or by the two-tailed unpaired t test using the InStat program (GraphPad). When in the box-and-whisker plots extends from the 25th to the 75th percentiles. , , a nonparametric test was warranted (due to nonnormality of data in Fig. 1), *p 0.05, **p 0.01 versus healthy control. a two-tailed Mann–Whitney U test was used. A p value <0.05 was taken as the level of signiﬁcance.

Results detected in periodontitis specimens (data not shown), as seen in http://www.jimmunol.org/ Increased APRIL and BLyS mRNA expression in periodontitis earlier reports (7, 37). To detect B cells/plasma cells, we stained k compared with health tissue specimens for Ig L chain, which was shown to be a reliable marker for B cells and terminally differentiated Ab-secreting plasma To investigate the expression pattern of APRIL (TNFSF13) and BLyS cells (30, 38). As a positive control, we used human tonsil speci- (TNFSF13B) and determine their possible association with peri- mens, which revealed the presence of immunopositive cells within odontitis, we obtained gingival biopsies from periodontally involved the subepithelial layer most likely representing a germinal center and healthy control individuals. Table I presents the mean demo- (Fig. 2A). All periodontally involved specimens examined presented graphic and clinical parameters of the two groups. There were no immunoreactivity for k L chain. Localization of k Lchain+ cells was significant differences in gender or age between periodontitis patients observed predominantly within connective tissue layers directly ad- by guest on September 28, 2021 and healthy control individuals, whereas, as expected, periodontally jacent to the gingival epithelium (Fig. 2B). No staining was observed involved sites exhibited significantly higher readings for probing for k L chain when FITC-conjugated nonimmune IgG was used in , depth than did control sites (p 0.01, Table I). APRIL and BLyS lieu of FITC-conjugated anti-human k L chain IgG (Fig. 2C). mRNA was detected in all gingival samples examined, although the In diseased samples stained for cell nuclei, k light chains, and relative expression of APRIL and BLyS was significantly higher in APRIL (Fig. 3), APRIL immunoreactivity was positive within the , diseased gingival samples than in controls (p 0.01,Fig.1).The gingival epithelium and underlying connective tissue adjoining the increased expression of APRIL and BLyS was comparable to that of basement membrane (Fig. 3C). In the gingival connective tissue, RANKL (TNFSF11) (Fig. 1), an osteoclastogenic cytokine that serves APRIL was detected dispersed in the extracellular matrix and as a biomarker for periodontal disease activity (36); conversely, the colocalized with Ig k L chain (yellow/orange staining) in cellular expression of osteoprotegerin (TNFRSF11b), a natural inhibitor of areas adjacent to the basement membrane (Fig. 3D, 3E). Similarly, RANKL (36), was significantly downregulated in diseased gingival BLyS was detected in diseased samples in the connective tissue , samples compared with healthy controls (p 0.05, Fig. 1). adjacent to the basement membrane (Fig. 4A–D). However, stronger Detection of Ig k L chain, APRIL, and BLyS protein in human BLyS immunostaining was observed in gingival granulomatous tis- gingiva sue (Fig. 4E–H). The variability of staining at different layers of the diseased gingival samples seemed to be related to the abundance of Analysis of H&E-stained sections (10 diseased and 2 controls) cell populations (infiltrating inflammatory cells and resident basal confirmed minimal inflammatory cell infiltration in healthy control epithelial cells). In contrast to diseased samples, control specimens specimens, whereas a moderate-to-heavy inflammatory infiltrate was displayed relatively poor staining for APRIL, BLyS, and Ig k L chain (Fig. 5). Detection of increased levels of APRIL and BLyS in gingival Table I. Demographic and clinical parameters of periodontally involved and healthy control groups tissue obtained from patients with chronic periodontitis prompted us to determine their functional significance relative to alveolar bone Parameters Health (n = 14) Periodontitis (n = 14) loss using an appropriate animal model. Age (y; mean 6 SD) 49.9 6 17.1 58.4 6 16.6 B cells are temporally and causally linked to alveolar bone loss Gender (n; male/female) 7/7 8/6 in experimental murine periodontitis Bleeding on probing No Yes Probing pocket depth 2.9 6 0.3 6.1 6 0.9* The ligature-induced periodontitis model involves placement of silk (mm; mean 6 SD) ligature around posterior teeth (e.g., the maxillary left second molar in Bone loss No Yes mice); this procedure causes local accumulation of bacteria and en- *p , 0.01 versus periodontally healthy. hances bacteria-mediated inflammation and bone loss at the ligated 1430 BLyS AND APRIL IN ALVEOLAR BONE LOSS

FIGURE 2. Ig k L chain staining of human mucosal tissues. (A) Immunohistochemical analysis of human tonsil shows diffuse staining of k L chain (green) within the connective tissue (CT) indicating the presence of B cells/plasma cells (red arrows). (B) Immunohistochemical analysis of human gingival tissue from a patient with chronic periodontitis shows prominent staining of k L chain (green) within the connective tissue adjacent to the basement membrane (red arrow). In both the tonsil (A) and the gingiva (B), occasional k L chain+ cells infiltrate the superficial epithelial (E) layer (yellow arrows). (C) Control staining of human gingival tissue from a patient with chronic periodontitis using FITC-conjugated nonimmune IgG. Nuclear staining (blue) by DAPI. Original magnification 3100. Scale bars, 100 mm.

site relative to the contralateral unligated site (“zero baseline”) (31, In stark contrast, WT mice developed signiﬁcantly more bone loss Downloaded from 33). In this model, there is robust complement activation and ex- thandidBcell–KOmiceatday10(p , 0.01,Fig.6C).Thesedata pression of high levels of IL-6 (35, 39), which are factors shown to suggest that B cells contribute to a late phase of ligature-induced promote B cell activation and differentiation (40, 41). Therefore, alveolar bone loss. Temporally, this occurred in parallel with the we reasoned that this may be an appropriate model to investigate the increase in the number of B-lineage cells within gingival connective role of B cells and their stimulatory cytokines APRIL and BLyS in tissue initiated 5 d after ligature placement.

periodontal bone loss. We first determined the time course of the http://www.jimmunol.org/ appearance of B cells/plasma cells in the murine gingiva following APRIL and BLyS are required for B cell/plasma cell initiation of periodontitis. Moreover, in view of conflicting results in involvement in alveolar bone loss the literature (18, 19), we examined whether the B-lineage cells are Consistent with our observations of elevated APRIL and BLyS in indeed involved in periodontal bone loss. human periodontitis, mice subjected to ligature-induced periodontitis B cells and plasma cells were readily detected in mouse gingiva 5 d displayed a gradual increase in APRIL and BLyS expression that after initiation of experimental periodontitis in WT, mice and their reached statistical significance after day 6 (p , 0.01,Fig.7A).In numbers increased in the following days (days 6–10, Fig. 6A). B cells/ contrast, RANKL expression was significantly upregulated as early as plasma cells were predominantly located in the connective tissue in day 5 (p , 0.01, Fig. 7A), consistent with the induction of B cell– proximity to the interface with the epithelium (Fig. 6A). Consistent independent bone loss at the same time interval (Fig. 6C). Consistent by guest on September 28, 2021 with this, IgG and IgM were more abundant at day 10 relative to day with the pattern of elevated APRIL and BLyS mRNA expression over 5 or earlier (Fig. 6B). WT mice displayed substantial bone loss by time, immunofluorescent staining of gingival tissue for APRIL and day 5, following a similar time course to that observed previously (35, BLyS protein revealed increased expression of these proteins at day 42). This bone loss in WT mice at day 5 was comparable to that 10 postligation compared with day 5 or the baseline (Fig. 7B). APRIL experienced by B cell–KO mice at the same time interval (Fig. 6C). staining seemed to be predominantly within the epithelium, consistent

FIGURE 3. APRIL expression in human periodontitis. Immunohistochemical analysis of diseased gingival tissue stained by DAPI to show cell nuclei (A), anti-k L chain mAb to show the presence of Ig-expressing/secreting cells (B cells/plasma cells) predominantly within the gingival connective tissue (CT) directly adjacent to the basement membrane (B), and anti-APRIL mAb to show immunoreactivity (yellow arrow) within the gingival epithelium (E) and underlying connective tissue adjacent to the basement membrane (C). (D) Fluorescence and differential interference contrast merged image showing positive colocalization (orange/yellow) of APRIL and k L chain expression within the gingival connective tissue. (E) Enlargement of the boxed area in (D) showing colocalized APRIL and k L chain surrounding DAPI-stained cells likely representing B cells/plasma cells. Scale bars, 100 mm. The Journal of Immunology 1431 Downloaded from FIGURE 4. BLyS expression in human periodontitis. Immunohistochemical analysis of diseased gingival tissue stained with DAPI to show cell nuclei (A), anti-Ig k L chain mAb to show the presence of Ig-expressing/secreting cells (B cells/plasma cells) predominantly within the gingival connective tissue (CT) (B), and anti-BLyS mAb to show immunoreactivity (yellow arrow) within the gingival connective tissue and, to a lesser extent, within the epithelium (E) (C). (D) Fluorescence and differential interference contrast merged image showing positive colocalization (orange) of BLyS and Ig k L chain expression within the gingival connective tissue. (E–H) Similar analysis as above using granulomatous tissue taken from the deepest aspect of an osseous defect. (E) DAPI staining to indicate cell nuclei. (F) Staining with anti-Ig k L chain mAb to indicate the presence of Ig-expressing/secreting cells (B cells/plasma cells).

(G) Staining with anti-BLyS mAb showing intense immunoreactivity (yellow arrows). (H) Fluorescence and differential interference contrast merged image http://www.jimmunol.org/ showing intense positive colocalization (orange) of BLyS and Ig k L chain at the heavily populated area of DAPI-stained cells likely representing B cells/ plasma cells. Original magnification 3200. Scale bars, 100 mm. with previous reports on the capacity of mucosal epithelia to produce as 100%) (Fig. 8C). Inhibition of alveolar bone loss mediated by the APRIL (43). In contrast, BLyS staining appeared to be compart- combined treatment with both mAbs was not significantly different mentalized within the connective tissue in close proximity to the from treatment with either mAb alone. Furthermore, bone loss in the epithelium (Fig. 7B). Although APRIL and BLyS colocalized par- presence of anti-APRIL and/or anti-BLyS was comparable to that seen tially, both proteins were detected at the boundaries of the epithelium in B cell–KO mice. Therefore, it appears that APRIL and BLyS are and the underlying connective tissue (coinciding with the basement each required for B cells to mediate a late phase of alveolar bone loss. by guest on September 28, 2021 membrane) (Fig. 7C), consistent with our observations in human To conclusively demonstrate that the observed effects of anti- gingival tissue. APRIL and anti-BLyS are mediated through action on B cells, the To determine whether APRIL and/or BLyS is involved in mediating following experiments were performed. First, in contrast to their alveolar bone loss, we microinjected the gingiva adjacent to ligated protective effects in WT mice, anti-APRIL and anti-BLyS lost their sites with specific neutralizing mAbs (i.e., that block binding of these ability to inhibit bone loss in B cell–KO mice (Fig. 8D). Second, using cytokines to their receptors) or isotype controls. Treatment with either the same protocol as in the bone loss study with WT mice (Fig. 8A), anti-APRIL mAb or anti-BLyS mAb caused comparable and signif- we showed that treatment with either anti-APRIL mAb or anti-BLyS icant inhibition of bone loss relative to their respective isotype controls mAb (but not with their isotype controls) diminished the number of (p , 0.01, Fig. 8A). Untreated mice (i.e., not microinjected at the B cells in the gingival tissue (p , 0.01, Fig. 9). Together, these data ligated sites) developed comparable bone loss to that experienced by show unequivocally that the involvement of APRIL and BLyS in isotype-control–treated mice (Fig. 8A). In a similar experiment, mi- ligature-induced bone loss requires the presence of B cells. croinjection of WT mice with a combination of anti-APRIL and anti- BLyS mAbs resulted in inhibition of bone loss relative to the com- Discussion bined isotype-control treatment (p , 0.01, Fig. 8B). The relative This study showed for the first time, to our knowledge, that the bone loss was calculated for each treatment in Fig. 8A and 8B and expression of APRIL and BLyS mRNA and protein is enhanced in compared with that observed in ligated B cell–KO and WT mice (set gingival tissue from patients with chronic periodontitis (relative to

FIGURE 5. APRIL and BLyS expression in healthy human gingival tissue. Immunohistochem- ical analysis of healthy gingiva stained for APRIL (A)orBLyS(B) (both red). The tissue was stained with DAPI to indicate cell nuclei (blue) and anti-Ig k L chain (green). The ﬂuorescent images (merged with differential interference contrast) show little, if any, positive staining for APRIL, BLyS, or Ig k L chain (compare with diseased tissue in Figs. 3 and 4). Original magniﬁcation 3100. Scale bars, 100 mm. CT, connective tissue; E, epithelium. 1432 BLyS AND APRIL IN ALVEOLAR BONE LOSS Downloaded from http://www.jimmunol.org/

FIGURE 6. Presence and impact of B cells in murine periodontitis. Tissue sections from ligature-induced periodontitis at the indicated time points were stained for CD19 (B cells; red) and CD138 (plasma cells; green) (A) or for mouse Igs, as indicated (B). All immunofluorescent sections in (A) and bottom panels in (B) were stained with DAPI to indicate cell nuclei. Original magnification 3200 (A), 3100 (B). Scale bars, 100 mm. In (A, top panel), H&E staining of the same tissue sections used for immunofluorescence analysis confirmed that the B-lineage cells are predominantly found in the connective tissue (CT) in proximity to the interface with the epithelium (E). In (A, bottom panel), B-lineage cells were enumerated from 50 random sections (10 from each of five mice/time point), and data are presented as box-and-whisker plots. The median is marked inside each box, and the ends of each box correspond by guest on September 28, 2021 to the interquartile range from the 25th to the 75th percentiles. *p , 0.01 versus D0. (C) Periodontal bone loss was induced in groups of WT or B cell–KO mice for 5 or 10 d by ligating a maxillary second molar and leaving the contralateral tooth unligated (baseline control). Data are mean 6 SD (n = 5 mice/ group). *p , 0.01 between the indicated groups. B, bone; T, tooth. samples from healthy individuals) and that Ab-mediated neutral- RANKL is produced by activated lymphocytes and stromal/ ization of APRIL and/or BLyS in experimental mouse periodon- osteoblastic cells (46) and is associated with periodontal disease ac- titis inhibits the late phase of alveolar bone loss that is mediated tivity in humans (36). Consistently, we detected increased RANKL by B-lineage cells. mRNA expression in diseased gingival specimens compared with APRIL immunostaining was detected in diseased human gingiva controls, thus correlating with the increased expression of APRIL within the epithelium and the directly adjacent connective tissue, and BLyS. Given that B cells constitute a major cellular source of where it colocalized with Ig k L chain. APRIL colocalization with k L RANKL in the bone resorptive lesions of human periodontitis (14), chain likely represents APRIL bound to either B cells or plasma cells. it is reasonable to suggest that APRIL and BLyS support the survival It was shown that intestinal epithelial cells secrete APRIL in response of RANKL-expressing B cells, thereby contributing to bone loss in to TLR-mediated stimulation by intestinal bacteria (43). Thus, it is the course of chronic periodontitis. Our intervention study (with likely that APRIL detected in the gingival epithelium was produced neutralizing mAbs to APRIL and/or BLyS) in the ligature-induced locally in response to dental plaque bacteria. An additional possibility periodontitis model is consistent with this hypothesis. Indeed, treat- is that APRIL might be systemically derived, in part, although the ment with either anti-APRIL mAb or anti-BLyS mAb diminished the serum levels of APRIL in patients with chronic periodontitis are not number of B cells in the gingival tissue and inhibited bone loss. significantly higher than those of healthy controls (44). The APRIL- Moreover, in contrast to their protective effects in WT mice, the rich zone within the gingiva was similar to that previously described treatments with anti-APRIL and anti-BLyS failed to inhibit bone loss in MALTs (24). The investigators showed that APRIL secretion is in B cell–KO mice, conclusively indicating that their effects require upregulated upon infection, and the cytokine is retained by heparan the presence of B cells. sulfate proteoglycans in subepithelial zones, thereby forming APRIL- In line with the human data, the expression of APRIL and BLyS also rich niches that promote the survival and accumulation of IgG- was upregulated in ligature-induced mouse periodontitis and corre- producing plasma cells (24). In contrast to APRIL, BLyS expression lated with elevated numbers of B cells/plasma cells. In this regard, the within the diseased gingiva was limited to the connective tissue. This induction of expression and release of APRIL and BLyS by stromal is consistent with the production of BLyS by myeloid cells, including and myeloid cells (e.g., epithelial cells and neutrophils, respectively) dendritic cells and macrophages (45). Nevertheless, similar to involves stimulation by several molecules, including G-CSF, LPS, and APRIL, BLyS was readily detected near the epithelial–connective other TLR ligands, C5a, and immune complexes (43, 47, 48), which tissue interface, an area rich in k L chain–expressing B-lineage cells. are present in inflamed gingiva in both humans and mice (4, 34, 35, The Journal of Immunology 1433

FIGURE 7. Expression of APRIL and BLyS in the murine gingival tissue. (A) Time course of APRIL (top panel), BLyS (middle panel), and RANKL (bottom panel) mRNA expression in the gingival tissue after ligature-induced periodontitis for 10 d in mice. Results were normalized to GAPDH mRNA and are presented relative to those at day 0, set as 1. Scatter dots and mean 6 SD were plotted for data from each group of mice (n = 5 mice/time point). (B) Gingival tissue sections from ligature-induced periodontitis at days 0, 5, and 10 (D0, D5, and D10) were stained for APRIL (upper panels)orBLyS(lower panels). The APRIL and BLyS ﬂuorescent images were merged with DAPI staining Downloaded from (blue) to reveal nuclei. (C) Staining for APRIL and BLyS in a gingival tissue section at day 10 postligation and merged image. Original magni- ﬁcation 3200. Scale bars, 100 mm. *p , 0.01 versus day 0. CT, connective tissue; E, epithelium. http://www.jimmunol.org/

49, 50). For the human study, our immunohistochemical observations (B cells, plasma cells, and memory T cells) associated with chronic of APRIL and BLyS were performed using keratinized human oral periodontitis (51). gingival explants. It is possible that the expression of these cytokines A recent cross-sectional study reported on the presence of APRIL maybehigherinthesulcularandjunctional epithelium, which are in and BLyS in the serum and saliva of patients with chronic periodontitis direct or closer contact with proinflammatory bacterial stimuli. It and healthy controls (44). Although BLyS was detected at signifi- should also be noted that, prior to collecting gingival tissue samples, cantly higher concentrations in both fluids from patients relative to the patients went through scaling and root planing, a procedure in- controls, there were no statistically significant differences in the levels by guest on September 28, 2021 volving the removal of the dental plaque biofilm. This clinical proof APRIL between the two groups (44). However, the levels of cedure might have contributed to decreased gingival inflammation cytokines in the gingiva, where they can be locally produced, are not and, hence, to an underestimation of APRIL and BLyS expression, necessarily correlated with their concentrations in serum or saliva. although this is not certain. In this regard, it was reported that scaling Rather, the gingival expression levels of the two cytokines might be and root planing do not significantly affect the inflammatory infiltrate better correlated with their concentrations in the gingival crevicular

FIGURE 8. Ab-mediated neutralization of APRIL or BLyS inhibits ligature-induced periodontal bone loss in a B cell–dependent manner. (A) Periodontal bone loss in WT mice locally microinjected or not with 5 mg anti-APRIL mAb or anti-BLyS mAb (or equal amounts of their isotype controls) 4 d after placing the ligatures and every day thereafter until the day before sacriﬁce (day 10). (B) Similar bone loss experiment in which mice were microinjected with a combination of anti-APRIL mAb and anti-BLyS mAb or a combination of their isotype controls (each mAb or control was used at 5 mg). (C) The bone loss for each treatment in (A)and(B) was calculated relative to their unligated controls (set as 100%). Also included for comparison is the bone loss of B cell– KO mice relative to WT controls from the experiment in Fig. 6C. (D) Periodontal bone loss in B cell–KO mice locally microinjected or not with 5 mg anti- APRIL mAb or anti-BLyS mAb (or equal amounts of their isotype controls) 4 d after placing the ligatures and every day thereafter until the day before sacriﬁce (day 10). Data are mean 6 SD (n = 5 mice/group). *p , 0.01 between indicated groups. 1434 BLyS AND APRIL IN ALVEOLAR BONE LOSS

FIGURE 9. Ab-mediated neutralization of APRIL or BLyS diminishes the numbers of B cells in the gingival tissue of mice subjected to ligature-induced Downloaded from periodontitis. (A) Mice undergoing ligature-induced periodontitis were locally microinjected or not (Untreated) with 5 mg anti-APRIL mAb or anti-BLyS mAb (or equal amounts of their isotype controls, IgG2b and IgG1, respectively) 4 d after placing the ligatures and every day thereafter until the day before sacriﬁce (day 10). Gingival tissue sections from these groups of mice, as well as from mice not subjected to ligature-induced periodontitis (Healthy gingiva), were stained for CD19 (B cells; red) and DAPI (blue) to reveal nuclei. Original magniﬁcation 3200. Scale bars, 100 mm. (B) B cells were enumerated from 30 random sections (10 from each of three mice/group), and data are presented as box-and-whisker plots. The median is marked inside each box, and the ends of each box correspond to the interquartile range from the 25th to the 75th percentiles. *p , 0.01 versus untreated and corresponding isotype control. B, bone; CT, connective tissue; E, epithelium. http://www.jimmunol.org/

fluid, which includes locally produced inflammatory mediators and in the advanced chronic lesion (7, 53). Therefore, B-lineage cells bathes the periodontal pockets. In a separate study, the same group likely play a more important role in the pathogenesis of human detected both APRIL and BLyS in the gingival crevicular fluid of periodontitis than in ligature-induced mouse periodontitis. There- periodontitis patients with or without systemic disease (rheumatoid fore, we think that treatment with anti-APRIL or anti-BLyS mAb arthritis or osteoporosis), although no comparisons were made with is likely to have a more pronounced therapeutic effect in human periodontally healthy controls (52). periodontitis than in the mouse model that we used to demonstrate Our data implicating B cells in periodontal bone loss in C57BL/6J proof of concept. Whether such therapies can have a strong pro- mice are consistent with an earlier study using a different model of tective effect in human periodontitis or whether they only confer by guest on September 28, 2021 mouse periodontitis (oral gavage) (19). Although the investigators a partial effect, representing a cotherapeutic possibility, can only associated the absence of B cells with protection from bone loss, be determined in future clinical trials. they did not monitor the appearance of B cells in WT C57BL/6J In summary, the elevated presence of APRIL and BLyS in B cell– mice (19). Taken together, our observational and interventional rich areas of chronically inflamed gingiva suggests that these cyto- studies showed that B cells and B cell–stimulatory cytokines are kines may contribute to bone loss by promoting the survival and temporally and causally linked to periodontal bone loss. Animal persistence of RANKL-expressing B cells/plasma cells. Support for models have limitations that do not allow investigators to faithfully this notion was provided by intervention experiments that implicated reproduce the complexity of a given human disease. However, dif- APRIL and BLyS in mediating B cell–dependent alveolar bone loss ferent aspects of the disease process can be represented by distinct in mice. Therefore, our study identified B cell–targeted approaches models, which can be used to test specific hypotheses, especially as potential accessory treatments for periodontitis. In this context, those that cannot be readily addressed in humans (27). By dem- several strategies targeting APRIL and/or BLyS are under clinical onstrating increased expression of APRIL and BLyS in the late development, and a neutralizing mAb to BLyS (belimumab) was stages of ligature-induced bone loss in mice, we had a relevant approvedbytheU.S.FoodandDrugAdministrationin2011for model to test the functional significance of the two cytokines. the treatment of systemic lupus erythematosus (23, 54, 55). Given Our results implicated both APRIL and BLyS in B cell–depen- that periodontitis is a prevalent disease, it would be interesting to dent bone loss; interestingly, neutralizing either cytokine was as determine the effects of systemic anti-APRIL/BLyS therapies on effective in blocking bone loss as their combined neutralization. periodontitis. However, dedicated, locally applied treatments with This suggests that B cells cannot mediate pathogenic bone loss these biologics in future clinical trials of periodontitis likely repre- in the absence of either of the two cytokines individually. This, sent safer and more effective therapeutic options. in turn, may facilitate a B cell–targeted therapeutic approach to treat periodontitis because it may not be necessary to neutralize Disclosures both cytokines at the same time. The authors have no financial conflicts of interest. Ligature-induced periodontitis in mice represents a model of ac- celerated disease that allowed us to observe the emergence of B cells and plasma cells in the late stages of the experimental bone loss References period. However, the model has limitations in that it does not rep- 1. Hajishengallis, G. 2015. Periodontitis: from microbial immune subversion to resent naturally occurring chronic periodontitis that could lead to the systemic inflammation. Nat. Rev. Immunol. 15: 30–44. establishment of B cell–dominated periodontitis lesions. Indeed, as 2. Lamont, R. J., and G. Hajishengallis. 2015. Polymicrobial synergy and dysbiosis in inflammatory disease. Trends Mol. Med. 21: 172–183. the severity of human periodontitis increases, the numbers of B cells 3. Hajishengallis, G. 2014. Immunomicrobial pathogenesis of periodontitis: key- and plasma cells also increase and become the predominant cell type stones, pathobionts, and host response. Trends Immunol. 35: 3–11. The Journal of Immunology 1435

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