TLR2 Signaling and Th2 Responses Drive Tannerella forsythia-Induced Periodontal Bone Loss This information is current as Srinivas R. Myneni, Rajendra P. Settem, Terry D. Connell, of October 4, 2021. Achsah D. Keegan, Sarah L. Gaffen and Ashu Sharma J Immunol 2011; 187:501-509; Prepublished online 1 June 2011; doi: 10.4049/jimmunol.1100683 http://www.jimmunol.org/content/187/1/501 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/06/01/jimmunol.110068 Material 3.DC1 http://www.jimmunol.org/ References This article cites 62 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/187/1/501.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 4, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology TLR2 Signaling and Th2 Responses Drive Tannerella forsythia-Induced Periodontal Bone Loss Srinivas R. Myneni,* Rajendra P. Settem,* Terry D. Connell,†,‡ Achsah D. Keegan,x Sarah L. Gaffen,{ and Ashu Sharma* Periodontal disease (PD) is a chronic inflammation of the tooth-supporting soft tissue and alveolar bone due to infection by a select group of Gram-negative microbes, which leads to tooth loss if untreated. Because mice deficient in CD4+ cells are resistant to infection-induced alveolar bone loss, Th cells have been implicated in bone-destructive processes during PD. However, the extent to which different Th cell subtypes play roles in pathogenesis or host protection remains to be defined and is likely to vary depending on the dominant microorganism involved. By far, Porphyromonas gingivalis is the best-studied periodontal microbe in PD. Although the Gram-negative anaerobe Tannerella forsythia is also a vital contributor to periodontal bone loss, almost nothing is known about immune responses to this organism. Previous studies from our laboratory revealed that T. forsythia induces Downloaded from periodontal bone loss in mice and that this bone loss depends on the bacterially expressed BspA protein. In this study, we showed that T. forsythia activates murine APCs primarily through TLR2-dependent signaling via BspA. Furthermore, T. forsythia in- fection causes a pronounced Th2 bias, evidenced by T cell expression of IL-5, but not IFN-g or IL-17, in draining lymph nodes. Consistently, deficiencies in TLR2 or STAT6 result in resistance to T. forsythia-induced alveolar bone loss. Thus, TLR2 signaling and Th2 cells play pathogenic roles in T. forsythia-induced alveolar bone destruction. The Journal of Immunology, 2011, 187: 501–509. http://www.jimmunol.org/ eriodontal disease (PD) is an inflammatory disease of the forsythia is a fastidious microbe with stringent growth require- tooth-supporting tissue (periodontium) that frequently ments. We were the first, to our knowledge, to document the vir- P leads to tooth loss (1) and is the most common cause of ulence potential of T. forsythia in a murine model of PD (9). In so inflammation-induced bone loss in humans. PD is caused by doing, we found that the alveolar bone loss is dependent on the a select group of anaerobic Gram-negative bacteria that colonize bacterially expressed virulence protein BspA (9). However, the subgingival spaces as biofilms. Periodontal bone destruction re- immune response to T. forsythia remains almost entirely undefined. sults mainly from the effects of the immune response to these Alveolar bone loss in response to oral infection by P. gingivalis biofilm bacteria (2, 3). The pathogenesis of PD is mediated by is dependent on the host response. For example, SCID mice or by guest on October 4, 2021 a polymicrobial consortium consisting of the Gram-negative mice specifically deficient in CD4+ T cells are resistant to alveolar pathogens Porphyromonas gingivalis, Treponema denticola, and bone loss due to P. gingivalis infection (10–12). Moreover, Th1 Tannerella forsythia, collectively known as the red complex (4–6). responses are associated with P. gingivalis-stimulated alveolar Although T. forsythia has been increasingly implicated in the bone loss in mice (13). In humans, the role of T cells in PD development and severity of PD, it remains a highly understudied pathobiology is complex, and data support a role for T cells in pathogen (7, 8). Its etiological role in PD has been recognized both protection and pathogenesis (6, 14–16). The existence of two relatively recently, and the virulence mechanisms of T. forsythia distinct effector CD4 T cell subsets has been recognized since are only just beginning to be defined (7). Unlike P. gingivalis, T. 1986 (17), with the description of Th1 (IFN-g–producing) and Th2 (IL-4-, IL-5- and IL-13–producing) cells. The Th17 lineage of IL-17–producing T cells was recognized in 2005 (18). In P. gin- *Department of Oral Biology, School of Dental Medicine, University at Buffalo, givalis infection, both Th1 and Th2 cytokines are found in the Buffalo, NY 14214; †Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214; ‡Witebsky Center for Microbial Pathogenesis and Im- periodontal lesion (6, 14–16). Prior to the discovery of Th17 cells, munology, University at Buffalo, Buffalo, NY 14214; xDepartment of Microbiology it was suggested that Th1 cells are characteristic of stable lesions, and Immunology, Center for Vascular and Inflammatory Diseases, University of whereas Th2 cells are associated with disease (14). However, IL- Maryland School of Medicine, Baltimore, MD 21201; and {Division of Rheumatol- ogy and Clinical Immunology, Department of Medicine, University of Pittsburgh, 17 was also documented in periodontitis patients with severe Pittsburgh, PA 15261 disease (19–22), and a significant number of CD4+ T cells isolated Received for publication March 8, 2011. Accepted for publication April 28, 2011. from gingival tissue of periodontitis patients express IL-17 (23). In This work was supported by U.S. Public Health R01 Grants DE1074905 (to A.S. P. gingivalis murine infections, IL-17 signaling is host protective and S.L.G.), DE14749 (to A.S.), DE13833 (to T.D.C.), and AI059775 and AI038985 by virtue of limiting infection via neutrophil mobilization (24). (to A.D.K.). The Th effector responses to T. forsythia are still poorly defined Address correspondence and reprint requests to Dr. Ashu Sharma, Department of and may be different from responses to P. gingivalis. This could Oral Biology, School of Dental Medicine, University at Buffalo, 311 Foster Hall, 3435 Main Street, Buffalo, NY 14214. E-mail address: [email protected] arise from differences in the nature of the pathogen-associated The online version of this article contains supplemental material. molecular patterns expressed on different periodontal pathogens, Abbreviations used in this article: ABC, alveolar bone crest; BMDC, bone marrow- which ultimately shape the adaptive response. In support of this derived dendritic cell; CEJ, cemento-enamel junction; cLN, cervical lymph node; notion, T. forsythia, unlike P. gingivalis, is unable to block neu- DC, dendritic cell; eLPS, Escherichia coli LPS; PD, periodontal disease; TRAP, trophil recruitment during infection in a mouse model (25), and tartrate-resistant acid phosphatase; WT, wild-type. recent studies demonstrated that T. forsythia, but not P. gingivalis, Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 preferentially activates TLR2. For instance, although P. gingivalis www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100683 502 TLR2/Th2 RESPONSES DRIVE T. FORSYTHIA-INDUCED BONE LOSS through its fimbriae and LPS predominantly activate TLR2 (26), detected using HRP-conjugated goat anti-mouse IgG (Bethyl Laboratories, whole bacteria (27) and both minor and major fimbrial proteins Montgomery, TX). Specific serum IgG isotype Ab was detected by addition activate TLR4/CD14/MD2 (28, 29). Thus, the role of CD4 ef- of biotinylated isotype-specific secondary Ab (rat anti-mouse IgG1 or IgG2a; Southern Biotechnology, Birmingham, AL) followed by streptavidin- fector responses in PD bone loss remains poorly defined (14, 30), conjugated HRP (Southern Biotechnology). ELISA wells were color de- particularly specific responses to T. forsythia. veloped with TMB Microwell enzyme substrate (Kirkgaards and Perry, TLRs can play protective or destructive roles, depending on the Gaithersburg, MD). After stopping the enzyme reaction by adding 0.1 N nature of the invading pathogen and its associated virulence de- H2SO4, plates were read at 495 nm. The titer was defined as the log2 of the highest dilution with a signal that was 0.1 OD units above background. terminants (31). T. forsythia, as well as its virulence factor BspA, induce proinflammatory cytokine and chemokine secretion through Isolation of dendritic cells and macrophages and stimulation TLR2 (32–34). Signaling by TLR2 in APCs and expression of Bone marrow-derived dendritic cells (BMDCs) were generated as described specific cytokines were suggested to favor Th2 responses (35–38). (41). Briefly, femurs and tibias were flushed with PBS. Erythrocytes were Moreover, the Th2-specific transcription factor STAT6 was linked lysed using M-Lyse buffer (R&D Systems, Minneapolis, MN), and cells were to susceptibility to PD in mice (39). Accordingly, it was compelling suspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, to determine the role of TLR2 and STAT6-mediated responses in T.