Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

The maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P. gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.

Dihydroceramides Derived from Bacteroidetes Species Sensitize TRPV1 Channels.

Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be sensitized under pro-inflammatory conditions. Bacterial membrane components, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharide (LPS), are released in the environment from the Gram-negative bacteria of the Bacteroidetes species colonizing the infected wounds. Here, we used intracellular calcium imaging and patch-clamp electrophysiology approaches to determine whether bacterially derived PEDHC, PGDHC, or LPS can modulate the activity of the TRPV1 channels heterologously expressed in HEK cells. We found that PEDHC and PGDHC can sensitize TRPV1 in a concentration-dependent manner, whereas LPS treatment does not significantly affect TRPV1 activity in HEK cells. We propose that sensitization of TRPV1 channels by Bacteroidetes-derived dihydroceramides may at least in part underlie the increased pain sensitivity associated with wound infections.

Effects of IL-34 and anti-IL-34 neutralizing mAb on alveolar bone loss in a ligature-induced model of periodontitis.

Macrophage colony-stimulating factor (M-CSF) and interleukin-34 (IL-34) are ligands for the colony-stimulating factor-1  receptor (CSF-1r) expressed on the surface of monocyte/macrophage lineage cells. The importance of coordinated signaling between M-CSF/receptor activator of the nuclear factor kappa-Β ligand (RANKL) in physiological and pathological bone remodeling and alveolar bone loss in response to oral bacterial colonization is well established. However, our knowledge about the IL-34/RANKL signaling in periodontal bone loss remains limited. Recently published cohort studies have demonstrated that the expression patterns of IL-34 are dramatically elevated in gingival crevicular fluid collected from patients with periodontitis. Therefore, the present study aims to evaluate the effects of IL-34 on osteoclastogenesis in vitro and in experimental ligature-mediated model of periodontitis using male mice. Our initial in vitro study demonstrated increased RANKL-induced osteoclastogenesis of IL-34-primed osteoclast precursors (OCPs) compared to M-CSF-primed OCPs. Using an experimental model of ligature-mediated periodontitis, we further demonstrated elevated expression of IL-34 in periodontal lesions. In contrast, M-CSF levels were dramatically reduced in these periodontal lesions. Furthermore, local injections of mouse recombinant IL-34 protein significantly elevated cathepsin K activity, increased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and promoted alveolar bone loss in periodontitis lesions. In contrast, anti-IL-34 neutralizing monoclonal antibody significantly reduced the level of alveolar bone loss and the number of TRAP-positive osteoclasts in periodontitis lesions. No beneficial effects of locally injected anti-M-CSF neutralizing antibody were observed in periodontal lesions. This study illustrates the role of IL-34 in promoting alveolar bone loss in periodontal lesions and proposes the potential of anti-IL34 monoclonal antibody (mAb)-based therapeutic regimens to suppress alveolar bone loss in periodontitis lesions.

IL-34 exacerbates pathogenic features of Alzheimer's disease and calvaria osteolysis in triple transgenic (3x-Tg) female mice.

Hallmark features of Alzheimer's disease (AD) include elevated accumulation of aggregated Aß40 and Aß42 peptides, hyperphosphorylated Tau (p-Tau), and neuroinflammation. Emerging evidence indicated that interleukin-34 (IL-34) contributes to AD and inflammatory osteolysis via the colony-stimulating factor-1 receptor (CSF-1r). In addition, CSF-1r is also activated by macrophage colony-stimulating factor-1 (M-CSF). While the role of M-CSF in bone physiology and pathology is well addressed, it remains controversial whether IL-34-mediated signaling promotes osteolysis, neurodegeneration, and neuroinflammation in relation to AD. In this study, we injected 3x-Tg mice with mouse recombinant IL-34 protein over the calvaria bone every other day for 42 days. Then, behavioral changes, brain pathology, and calvaria osteolysis were evaluated using various behavioral maze and histological assays. We demonstrated that IL-34 administration dramatically elevated AD-like anxiety and memory loss, pathogenic amyloidogenesis, p-Tau, and RAGE expression in female 3x-Tg mice. Furthermore, IL-34 delivery promoted calvaria inflammatory osteolysis compared to the control group. In addition, we also compared the effects of IL-34 and M-CSF on macrophages, microglia, and RANKL-mediated osteoclastogenesis in relation to AD pathology in vitro. We observed that IL-34-exposed SIM-A9 microglia and 3x-Tg bone marrow-derived macrophages released significantly elevated amounts of pro-inflammatory cytokines, TNF-α, IL-1ß, and IL-6, compared to M-CSF treatment in vitro. Furthermore, IL-34, but not M-CSF, elevated RANKL-primed osteoclastogenesis in the presence of Aß40 and Aß42 peptides in bone marrow derived macrophages isolated from female 3x-Tg mice. Collectively, our data indicated that IL-34 elevates AD-like features, including behavioral changes and neuroinflammation, as well as osteoclastogenesis in female 3x-Tg mice.