Quercetin Preserves Oral Cavity Health by Mitigating Inflammation and Microbial Dysbiosis.

Failure to attenuate inflammation coupled with consequent microbiota changes drives the development of bone-destructive periodontitis. Quercetin, a plant-derived polyphenolic flavonoid, has been linked with health benefits in both humans and animals. Using a systematic approach, we investigated the effect of orally delivered Quercetin on host inflammatory response, oral microbial composition and periodontal disease phenotype. In vivo, quercetin supplementation diminished gingival cytokine expression, inflammatory cell infiltrate and alveolar bone loss. Microbiome analyses revealed a healthier oral microbial composition in Quercetin-treated versus vehicle-treated group characterized by reduction in the number of pathogenic species including Enterococcus, Neisseria and Pseudomonas and increase in the number of non-pathogenic Streptococcus sp. and bacterial diversity. In vitro, Quercetin diminished inflammatory cytokine production through modulating NF-κB:A20 axis in human macrophages following challenge with oral bacteria and TLR agonists. Collectively, our findings reveal that Quercetin supplement instigates a balanced periodontal tissue homeostasis through limiting inflammation and fostering an oral cavity microenvironment conducive of symbiotic microbiota associated with health. This proof of concept study provides key evidence for translational studies to improve overall health.

A20 Restricts Inflammatory Response and Desensitizes Gingival Keratinocytes to Apoptosis.

The pathophysiology of periodontal disease involves a perturbed immune system to a dysbiotic microflora leading to unrestrained inflammation, collateral tissue damage, and various systemic complications. Gingival epithelial cells function as an important part of immunity to restrict microbial invasion and orchestrate the subsequent innate responses. A20 (TNFAIP3), an ubiquitin-editing enzyme, is one of the key regulators of inflammation and cell death in numerous tissues including gastrointestinal tract, skin, and lungs. Emerging evidence indicates A20 as an essential molecule in the oral mucosa as well. In this study, we characterized the role of A20 in human telomerase immortalized gingival keratinocytes (TIGKs) through loss and gain of function assays in preclinical models of periodontitis. Depletion of A20 through gene editing in TIGKs significantly increased IL-6 and IL-8 secretion in response to Porphyromonas gingivalis infection while A20 over-expression dampened the cytokine production compared to A20 competent cells through modulating NF-κB signaling pathway. In the subsequent experiments which assessed apoptosis, A20 depleted TIGKs displayed increased levels of cleaved caspase 3 and DNA fragmentation following P. gingivalis infection and TNF/CHX challenge compared to A20 competent cells. Consistently, there was reduced apoptosis in the cells overexpressing A20 compared to the control cells expressing GFP further substantiating the role of A20 in regulating gingival epithelial cell fate in response to exogenous insult. Collectively, our findings reveal first systematic evidence and demonstrate that A20 acts as a regulator of inflammatory response in gingival keratinocytes through its effect on NF-κB signaling and desensitizes cells to bacteria and cytokine induced apoptosis in the oral mucosa. As altered A20 levels can have profound effect on different cellular responses, future studies will determine whether A20-targeted therapies can be exploited to restrain periodontal inflammation and maintain oral mucosa tissue homeostasis.

A20 Orchestrates Inflammatory Response in the Oral Mucosa through Restraining NF-κB Activity.

Deregulated immune response to a dysbiotic resident microflora within the oral cavity leads to chronic periodontal disease, local tissue destruction, and various systemic complications. To preserve tissue homeostasis, inflammatory signaling pathways involved in the progression of periodontitis must be tightly regulated. A20 (TNFAIP3), a ubiquitin-editing enzyme, has emerged as one of the key regulators of inflammation. Yet, the function of A20 in the oral mucosa and the biological pathways in which A20 mitigates periodontal inflammation remain elusive. Using a combination of in vivo and ex vivo disease models, we report in this study that A20 regulates inflammatory responses to a keystone oral bacterium, Porphyromonas gingivalis, and restrains periodontal inflammation through its effect on NF-κB signaling and cytokine production. Depletion of A20 using gene editing in human macrophage-like cells (THP-1) significantly increased cytokine secretion, whereas A20 overexpression using lentivirus infection dampened the cytokine production following bacterial challenge through modulating NF-κB activity. Similar to human cells, bone marrow-derived macrophages from A20-deficient mice infected with P. gingivalis displayed increased NF-κB activity and cytokine production compared with the cells isolated from A20-competent mice. Subsequent experiments using a murine ligature-induced periodontitis model showed that even a partial loss of A20 promotes an increased inflammatory phenotype and more severe bone loss, further verifying the critical function of A20 in the oral mucosa. Collectively, to our knowledge, these findings reveal the first systematic evidence of a physiological role for A20 in the maintenance of oral tissue homeostasis as a negative regulator of inflammation.

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

Toll-like receptor 9 (TLR9)-deficient (TLR9-/-) 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-/- 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-/- ligated mice compared to the unligated controls. The peritoneal infection model using Porphyromonas gingivalis, a keystone pathogen for periodontitis, revealed reduced neutrophils in TLR9-/- mice on day 1 postinfection compared to the levels in WT mice. Transcriptomics analyses showed increased expression of A20 (tumor necrosis factor alpha [TNF-α]-induced protein 3 [TNFAIP3]), an inhibitor of the NF-κB pathway and a negative regulator of TLR signaling, in ligated TLR9-/- mouse gingival tissues compared to its expression in the WT. Ex vivo, TLR9-/- 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.

Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division.

Polarized segregation of proteins in T cells is thought to play a role in diverse cellular functions including signal transduction, migration, and directed secretion of cytokines. Persistence of this polarization can result in asymmetric segregation of fate-determining proteins during cell division, which may enable a T cell to generate diverse progeny. Here, we provide evidence that a lineage-determining transcription factor, T-bet, underwent asymmetric organization in activated T cells preparing to divide and that it was unequally partitioned into the two daughter cells. This unequal acquisition of T-bet appeared to result from its asymmetric destruction during mitosis by virtue of concomitant asymmetric segregation of the proteasome. These results suggest a mechanism by which a cell may unequally localize cellular activities during division, thereby imparting disparity in the abundance of cell fate regulators in the daughter cells.

Cutting edge: Lymphoproliferation caused by Fas deficiency is dependent on the transcription factor eomesodermin.

A hallmark of autoimmune lymphoproliferative syndrome (ALPS), caused by mutation of the Fas death receptor, is massive lymphadenopathy from aberrant expansion of CD4(-)CD8(-) (double-negative [DN]) T cells. Eomesodermin (Eomes) is a member of the T-box family of transcription factors and plays critical roles in effector cell function and memory cell fitness of CD8(+) T lymphocytes. We provide evidence in this study that DN T cells exhibit dysregulated expression of Eomes in humans and mice with ALPS. We also find that T cell-specific deletion of Eomes prevents lymphoid hypertrophy and accumulation of DN T cells in Fas-mutant mice. Although Eomes has critical physiological roles in the function and homeostasis of CD8(+) T cells, overexpression of Eomes appears to enable pathological induction or expansion of unusual CD8-related T cell subsets. Thus, antagonism of Eomes emerges as a therapeutic target for DN T cell ablation in ALPS.

Cutting edge: The transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche.

CD8(+) T cells responding to intracellular infection give rise to cellular progeny that become terminally differentiated effector cells and self-renewing memory cells. T-bet and eomesodermin (Eomes) are key transcription factors of cytotoxic lymphocyte lineages. We show in this study that CD8(+) T cells lacking Eomes compete poorly in contributing to the pool of Ag-specific central memory cells. Eomes-deficient CD8(+) T cells undergo primary clonal expansion but are defective in long-term survival, populating the bone marrow niche and re-expanding postrechallenge. The phenotype of Eomes-deficient CD8(+) T cells supports the hypothesis that T-bet and Eomes can act redundantly to induce effector functions, but can also act to reciprocally promote terminal differentiation versus self-renewal of Ag-specific memory cells.