BET bromodomain inhibitors suppress inflammatory activation of gingival fibroblasts and epithelial cells from periodontitis patients

BET bromodomain proteins are important epigenetic regulators of gene expression that bind acetylated histone tails and regulate the formation of acetylation-dependent chromatin complexes. BET inhibitors suppress inflammatory responses in multiple cell types and animal models, and protect against bone loss in experimental periodontitis in mice. Here, we analyzed the role of BET proteins in inflammatory activation of gingival fibroblasts (GFs) and gingival epithelial cells (GECs). We show that the BET inhibitors I-BET151 and JQ1 significantly reduced expression and/or production of distinct, but overlapping, profiles of cytokine-inducible mediators of inflammation and bone resorption in GFs from healthy donors (IL6, IL8, IL1B, CCL2, CCL5, COX2, and MMP3) and the GEC line TIGK (IL6, IL8, IL1B, CXCL10, MMP9) without affecting cell viability. Activation of mitogen-activated protein kinase and nuclear factor-κB pathways was unaffected by I-BET151, as was the histone acetylation status, and new protein synthesis was not required for the anti-inflammatory effects of BET inhibition. I-BET151 and JQ1 also suppressed expression of inflammatory cytokines, chemokines, and osteoclastogenic mediators in GFs and TIGKs infected with the key periodontal pathogen Porphyromonas gingivalis. Notably, P. gingivalis internalization and intracellular survival in GFs and TIGKs remained unaffected by BET inhibitors. Finally, inhibition of BET proteins significantly reduced P. gingivalis-induced inflammatory mediator expression in GECs and GFs from patients with periodontitis. Our results demonstrate that BET inhibitors may block the excessive inflammatory mediator production by resident cells of the gingival tissue and identify the BET family of epigenetic reader proteins as a potential therapeutic target in the treatment of periodontal disease

Association of distinct fine specificities of anti-citrullinated peptide antibodies with elevated immune responses to Prevotella intermedia in a subgroup of patients with rheumatoid arthritis and periodontitis

Objective In addition to the long-established link with smoking, periodontitis (PD) is also a risk factor for rheumatoid arthritis (RA). To elucidate the mechanism by which PD could induce antibodies to citrullinated peptides (ACPA), we examine the antibody response to a novel citrullinated peptide from cytokeratin type I 13 identified in gingival crevicular fluid (GCF), and compare the response to 4 other citrullinated peptides in patients with RA, well-characterized for PD and smoking. Methods The citrullinomes of GCF and periodontal tissue from people with PD were mapped by mass spectrometry. Antibodies to citrullinated peptides from cytokeratin type I 13 (cCK13), tenascin-C (cTNC5), vimentin (cVIM), enolase (CEP-1) and fibrinogen β (cFIBβ) were examined by ELISA in patients with RA (n=287) and osteoarthritis (OA) (n=330), and cross-reactivity assessed by inhibition assays. Results A novel citrullinated peptide cCK13-1 (444TSNASGR-cit-TSDV-cit-RP458) identified in GCF, exhibited elevated antibody responses in RA patients (24%). Anti-cCK13-1 antibodies correlated with anti-cTNC5 antibodies, and absorption experiments confirmed this was not due to cross-reactivity. Only anti-cCK13-1 and anti-cTNC5 were associated with antibodies to the periodontal pathogen Prevotella intermedia (p=0.05 and p =0.001 respectively), but not with antibodies to Porphyromonas gingivalis arginine gingipains. Antibodies to CEP-1, cFIBβ and cVIM correlated with each other, and with smoking and shared epitope risk factors in RA. Conclusion This study identifies two groups of ACPA fine specificities associated with different RA risk factors; one predominantly linked to smoking and shared epitope, the other linking anti- cTNC5 and cCK13-1 to infection with the periodontal pathogen P. intermedia

Methods designed for the identification and characterization of in vitro and in vivo chromatin assembly mutants in Saccharomyces cerevisiae

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae. Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5. Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that the in vitro and in vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks

GLRB allelic variation associated with agoraphobic cognitions, increased startle response and fear network activation : a potential neurogenetic pathway to panic disorder

The molecular genetics of panic disorder (PD) with and without agoraphobia (AG) are still largely unknown and progress is hampered by small sample sizes. We therefore performed a genome-wide association study with a dimensional, PD/AG - related anxiety phenotype based on the Agoraphobia Cognition Questionnaire (ACQ) in a sample of 1,370 healthy German volunteers of the CRC TRR58 MEGA study wave 1. A genome-wide significant association was found between ACQ and single non-coding nucleotide variants of the GLRB gene (rs78726293, p=3.3x10-8; rs191260602, p=3.9x10-8). We followed up on this finding in a larger dimensional ACQ sample (N=2,547) and in independent samples with a dichotomous AG phenotype based on the Symptoms Checklist (SCL-90; N=3,845) and a case control sample with the categorical phenotype PD/AG (Ncombined =1,012) obtaining highly significant p-values also for GLRB single nucleotide variants rs17035816 (p=3.8x10-4) and rs7688285 (p=7.6x10-5). GLRB gene expression was found to be modulated by rs7688285 in brain tissue as well as cell culture. Analyses of intermediate PD/AG phenotypes demonstrated increased startle reflex and increased fear network as well as general sensory activation by GLRB risk gene variants rs78726293, rs191260602, rs17035816 and rs7688285. Partial Glrb knockout-mice demonstrated an agoraphobic phenotype. In conjunction withthe clinical observation that rare coding GLRB gene mutations are associated with the neurological disorder hyperekplexia characterized by a generalized startle reaction and agoraphobic behavior, our data provide evidence that non-coding, though functional GLRB gene polymorphisms may predispose to PD by increasing startle response and agoraphobic cognitions.PostprintPeer reviewe