Antimicrobial-induced oral dysbiosis exacerbates naturally occurring alveolar bone loss.

Periodontitis-mediated alveolar bone loss is caused by dysbiotic shifts in the commensal oral microbiota that upregulate proinflammatory osteoimmune responses. The study purpose was to determine whether antimicrobial-induced disruption of the commensal microbiota has deleterious effects on alveolar bone. We administered an antibiotic cocktail, minocycline, or vehicle-control to sex-matched C57BL/6T mice from age 6- to 12 weeks. Antibiotic cocktail and minocycline had catabolic effects on alveolar bone in specific-pathogen-free (SPF) mice. We then administered minocycline or vehicle-control to male mice reared under SPF and germ-free conditions, and we subjected minocycline-treated SPF mice to chlorhexidine oral antiseptic rinses. Alveolar bone loss was greater in vehicle-treated SPF versus germ-free mice, demonstrating that the commensal microbiota drives naturally occurring alveolar bone loss. Minocycline- versus vehicle-treated germ-free mice had similar alveolar bone loss outcomes, implying that antimicrobial-driven alveolar bone loss is microbiota dependent. Minocycline induced phylum-level shifts in the oral bacteriome and exacerbated naturally occurring alveolar bone loss in SPF mice. Chlorhexidine further disrupted the oral bacteriome and worsened alveolar bone loss in minocycline-treated SPF mice, validating that antimicrobial-induced oral dysbiosis has deleterious effects on alveolar bone. Minocycline enhanced osteoclast size and interface with alveolar bone in SPF mice. Neutrophils and plasmacytoid dendritic cells were upregulated in cervical lymph nodes of minocycline-treated SPF mice. Paralleling the upregulated proinflammatory innate immune cells, minocycline therapy increased TH 1 and TH 17 cells that have known pro-osteoclastic actions in the alveolar bone. This report reveals that antimicrobial perturbation of the commensal microbiota induces a proinflammatory oral dysbiotic state that exacerbates naturally occurring alveolar bone loss.

Epigenetic Reexpression of Hemoglobin F Using Reversible LSD1 Inhibitors: Potential Therapies for Sickle Cell Disease.

Sickle cell disease (SCD) is caused by a single nucleotide polymorphism on chromosome 11 in the ß-globin gene. The resulting mutant hemoglobin S (HbS) is a poor oxygen transporter and causes a variety of vascular symptoms and organ failures. At birth, the DRED epigenetic complex forms and silences the γ-globin gene, and fetal hemoglobin (HbF, 2 α-, and 2 γ-subunits) is replaced by adult HbA (α2ß2) or HbS (α2ßs 2) in SCD patients. HbF is a potent inhibitor of HbS polymerization, thus alleviating the symptoms of SCD. The current therapy, hydroxyurea (HU), increases γ-globin and the HbF content in sickle cells but is highly underutilized due to concern for adverse effects and other complications. The DRED complex contains the epigenetic eraser lysine-specific demethylase 1 (LSD1), which appears to serve as a scaffolding protein. Our recently discovered 1,2,4-triazole derivatives and cyclic peptide LSD1 inhibitors promote the upregulation of γ-globin production in vitro without significant toxicity. Herein, we demonstrate that these LSD1 inhibitors can be used to disrupt the DRED complex and increase the cellular HbF content in vitro and in vivo. This approach could lead to an innovative and effective treatment for SCD.

Specific Commensal Bacterium Critically Regulates Gut Microbiota Osteoimmunomodulatory Actions During Normal Postpubertal Skeletal Growth and Maturation.

The commensal gut microbiota critically regulates immunomodulatory processes that influence normal skeletal growth and maturation. However, the influence of specific microbes on commensal gut microbiota osteoimmunoregulatory actions is unknown. We have shown previously that the commensal gut microbiota enhances TH17/IL17A immune response effects in marrow and liver that have procatabolic/antianabolic actions in the skeleton. Segmented filamentous bacteria (SFB), a specific commensal gut bacterium within phylum Firmicutes, potently induces TH17/IL17A-mediated immunity. The study purpose was to delineate the influence of SFB on commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal development. Two murine models were utilized: SFB-monoassociated mice versus germ-free (GF) mice and specific-pathogen-free (SPF) mice +/- SFB. SFB colonization was validated by 16S rDNA analysis, and SFB-induced TH17/IL17A immunity was confirmed by upregulation of Il17a in ileum and IL17A in serum. SFB-colonized mice had an osteopenic trabecular bone phenotype, which was attributed to SFB actions suppressing osteoblastogenesis and enhancing osteoclastogenesis. Intriguingly, SFB-colonized mice had increased expression of proinflammatory chemokines and acute-phase reactants in the liver. Lipocalin-2 (LCN2), an acute-phase reactant and antimicrobial peptide, was substantially elevated in the liver and serum of SFB-colonized mice, which supports the notion that SFB regulation of commensal gut microbiota osteoimmunomodulatory actions are mediated in part through a gut-liver-bone axis. Proinflammatory TH17 and TH1 cells were increased in liver-draining lymph nodes of SFB-colonized mice, which further substantiates that SFB osteoimmune-response effects may be mediated through the liver. SFB-induction of Il17a in the gut and Lcn2 in the liver resulted in increased circulating levels of IL17A and LCN2. Recognizing that IL17A and LCN2 support osteoclastogenesis/suppress osteoblastogenesis, SFB actions impairing postpubertal skeletal development appear to be mediated through immunomodulatory effects in both the gut and liver. This research reveals that specific microbes critically impact commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal growth and maturation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Dual inhibitors of LSD1 and spermine oxidase.

We have previously described the synthesis and evaluation of 3,5-diamino-1,2,4-triazole analogues as inhibitors of the flavin-dependent histone demethylase LSD1. These compounds are potent inhibitors of LSD1 without activity against monoamine oxidases A and B, and promote the elevation of H3K4me2 levels in tumor cells in vitro. We now report that the cytotoxicity of these analogues in pancreatic tumor cells correlates with the overexpression of LSD1 in each tumor type. In addition, we show that a subset of these 3,5-diamino-1,2,4-triazole analogues inhibit a related flavin-dependent oxidase, the polyamine catabolic enzyme spermine oxidase (SMOX) in vitro.

Antibiotic Perturbation of Gut Microbiota Dysregulates Osteoimmune Cross Talk in Postpubertal Skeletal Development.

Commensal gut microbiota-host immune responses are experimentally delineated via gnotobiotic animal models or alternatively by antibiotic perturbation of gut microbiota. Osteoimmunology investigations in germ-free mice, revealing that gut microbiota immunomodulatory actions critically regulate physiologic skeletal development, highlight that antibiotic perturbation of gut microbiota may dysregulate normal osteoimmunological processes. We investigated the impact of antibiotic disruption of gut microbiota on osteoimmune response effects in postpubertal skeletal development. Sex-matched C57BL/6T mice were administered broad-spectrum antibiotics or vehicle-control from the age of 6 to 12 weeks. Antibiotic alterations in gut bacterial composition and skeletal morphology were sex dependent. Antibiotics did not influence osteoblastogenesis or endochondral bone formation, but notably enhanced osteoclastogenesis. Unchanged Tnf or Ccl3 expression in marrow and elevated tumor necrosis factor-α and chemokine (C-C motif) ligand 3 in serum indicated that the pro-osteoclastic effects of the antibiotics are driven by increased systemic inflammation. Antibiotic-induced broad changes in adaptive and innate immune cells in mesenteric lymph nodes and spleen demonstrated that the perturbation of gut microbiota drives a state of dysbiotic hyperimmune response at secondary lymphoid tissues draining local gut and systemic circulation. Antibiotics up-regulated the myeloid-derived suppressor cells, immature myeloid progenitor cells known for immunosuppressive properties in pathophysiologic inflammatory conditions. Myeloid-derived suppressor cell-mediated immunosuppression can be antigen specific. Therefore, antibiotic-induced broad suppression of major histocompatibility complex class II antigen presentation genes in bone marrow discerns that antibiotic perturbation of gut microbiota dysregulates critical osteoimmune cross talk.

Inhibition of the histone demethylase KDM4B leads to activation of KDM1A, attenuates bacterial-induced pro-inflammatory cytokine release, and reduces osteoclastogenesis.

Periodontal disease (PD) afflicts 46% of Americans with no effective adjunctive therapies available. While most pharmacotherapy for PD targets bacteria, the host immune response is responsible for driving tissue damage and bone loss in severe disease. Herein, we establish that the histone demethylase KDM4B is a potential drug target for the treatment of PD. Immunohistochemical staining of diseased periodontal epithelium revealed an increased abundance of KDM4B that correlates with inflammation. In murine calvarial sections exposed to Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa-LPS), immunohistochemical staining revealed a significant increase in KDM4B protein expression. The 8-hydroxyquinoline ML324 is known to inhibit the related demethylase KDM4E in vitro, but has not been evaluated against any other targets. Our studies indicate that ML324 also inhibits KDM4B (IC50: 4.9 µM), and decreases the pro-inflammatory cytokine response to an Aa-LPS challenge in vitro. Our results suggest that KDM4B inhibition-induced immunosuppression works indirectly, requiring new protein synthesis. In addition, fluorescence-stained macrophages exhibited a significant decrease in global monomethyl histone 3 lysine 4 (H3K4me) levels following an Aa-LPS challenge that was prevented by KDM4B inhibition, suggesting this effect is produced through KDM1A-mediated demethylation of H3K4. Finally, ML324 inhibition of KDM4B in osteoclast progenitors produced a significant reduction in Aa-LPS-induced osteoclastogenesis. These data link histone methylation with host immune response to bacterial pathogens in PD, and suggest a previously unreported, alternative mechanism for epigenetic control of the host inflammatory environment. As such, KDM4B represents a new therapeutic target for treating hyper-inflammatory diseases that result in bone destruction.