Interplay of Toll-Like Receptor 9, Myeloid Cells, and Deubiquitinase A20 in Periodontal Inflammation

Abstract

Toll-like receptor 9 (TLR9)-deficient (TLR9 Ϫ/Ϫ ) mice are resistant to periodontitis, a disease characterized by a dysbiotic microbiota and deregulated immune response and resulting in tooth loss and various systemic conditions. However, the mechanisms and biological pathways by which TLR9 instigates periodontal inflammation are yet to be identified. In a ligature-induced model of periodontitis, we demonstrate that TLR9 Ϫ/Ϫ mice exhibited significantly less alveolar bone loss than their wild-type (WT) counterparts. Consistent with the disease phenotype, gingival tissues showed significantly more inflammatory cell infiltration in the WT ligated but not in the TLR9 Ϫ/Ϫ ligated mice compared to the unligated controls. The peritoneal infection model using Porphyromonas gingivalis, a keystone pathogen for periodontitis, revealed reduced neutrophils in TLR9 Ϫ/Ϫ mice on day 1 postinfection compared to the levels in WT mice. Transcriptomics analyses showed increased expression of A20 (tumor necrosis factor alpha [TNF-␣]-induced protein 3 [TNFAIP3]), an inhibitor of the NF-B pathway and a negative regulator of TLR signaling, in ligated TLR9 Ϫ/Ϫ mouse gingival tissues compared to its expression in the WT. Ex vivo, TLR9 Ϫ/Ϫ bone marrow-derived macrophages produced more A20 than WT cells following P. gingivalis challenge. Clinically, A20 was modestly upregulated in human gingival tissue specimens from chronic periodontitis patients, further confirming the biological relevance of A20 in periodontal inflammation. We conclude that TLR9 modulates periodontal disease progression at both the cellular and molecular level and identify A20 as a novel downstream signaling molecule in the course of periodontal inflammation. Understanding the regulation of the TLR9 signaling pathway and the involvement of A20 as a limiting factor of inflammation will uncover alternative therapeutic targets to treat periodontitis and other chronic inflammatory diseases.

Assessment of microRNA‐144‐5p and its putative targets in inflamed gingiva from chronic periodontitis patients

“…The genes encoding important molecules involved in periodontal inflammation identified in previously published articles include matrix metallopeptidases including MMP1 , MMP2 , MMP3 , MMP8 , MMP9 and MMP13 and tissue inhibitors of metalloproteinases including TIMP1 , TIMP3 and TIMP4 ; various interleukin (IL) families including IL1 β, IL2 , IL4 , IL6 , IL8 , IL10 , IL11 , IL 12 , IL17A , IL17F , IL18 , IL21 , IL23 and IL35 ; the pentraxin family including C‐reactive protein/pentraxin 1 ( CRP / PTX1 ) and pentraxin 3 ( PTX3 ); the TNF superfamily TNF‐ α and the receptor activator of the NFκB ligand/TNF superfamily member 11 ( RANKL / TNFSF11 ); the TNF receptor superfamily osteoprotegerin/TNF receptor superfamily member 11b ( OPG / TNFRSF11B ); the transforming growth factor (TGF) superfamily TGF β 1 ; the TLR family including TLR2 and TLR4 ; chemokines including C‐C motif chemokine ligands ( CCL2 , CCL3 , CCL5 , CCL19 , CCL20 ), C‐C motif chemokine receptor 4 ( CCR4 ) and C‐X‐C motif chemokine ligands ( CXCL6 , CXCL8 / IL8 ); suppressors of cytokine signalling including SOCS1 and SOCS3 ; interferon IFN‐ γ; transcription regulator T‐box 21 ( TBX21 ); NF κ B / NFKB1 ; TNF‐α‐induced protein 3 ( TNFAIP3 ); peptidylprolyl isomerase A/cyclophilin A ( PPIA / CypA ); and cyclooxygenase 2/prostaglandin‐endoperoxide synthase 2 ( COX2/PTGS2 ) . By combining the retrieval and computer algorithm prediction results, potential target genes related to periodontal inflammation were determined.…”

“…NFκB/NFKB1 36 ; TNFα-induced protein 3 (TNFAIP3) 37 ; peptidylprolyl isomerase A/cyclophilin A (PPIA/CypA) 38 ; and cyclooxygenase 2/prostaglandin-endoperoxide synthase 2 (COX2/PTGS2). 39 By combining the retrieval and computer algorithm prediction results, potential target genes related to periodontal inflammation were determined.…”

A20 Restricts Inflammatory Response and Desensitizes Gingival Keratinocytes to Apoptosis

“…A20 is implicated as a key regulator of epithelial cell biology in a variety of tissues (13)(14)(15)(16)(17)(18)(19). Clinically, A20 protein is expressed in the gingival epithelium, but there are still no studies investigating its function in the gingival epithelial cells (20). In this study, through elaborate use of gain and loss of function assays, we identified A20 as one of the novel regulators of inflammatory and apoptotic responses in gingival keratinocytes.…”

“…Porphyromonas gingivalis (strain ATCC33277) was maintained in Brain Heart Infusion supplemented with 0.05% Yeast Extract, 5 g/mL Hermin, 0.5 g/mL Vitamin K and 0.1% cysteine. The culture was placed in anaerobic chambers at 37 • C. Bacteria were killed by heating at 80 • C for 10 min (20). Heat-killed bacteria were used for all experiments except for P. gingivalis induced apoptosis.…”

“…Cas9-screening-s GAGGTGCTAAGATCTTTTGCCTACAGAC Cas9-screening-as GTAAAGGCCATTGGCAGAAC protocol as described (7,20). Briefly, after SDS-PAGE, the proteins were transferred to PVDF membranes (Millipore).…”