Transcriptome Analysis of B Cell Immune Functions in Periodontitis: Mucosal Tissue Responses to the Oral Microbiome in Aging

Evidence has shown activation of T and B cells in gingival tissues in experimental models and in humans diagnosed with periodontitis. The results of this adaptive immune response are noted both locally and systemically with antigenic specificity for an array of oral bacteria, including periodontopathic species, e.g., Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. It has been recognized through epidemiological studies and clinical observations that the prevalence of periodontitis increases with age. This report describes our studies evaluating gingival tissue transcriptomes in humans and specifically exploiting the use of a non-human primate model of naturally occurring periodontitis to delineate gingival mucosal tissue gene expression profiles focusing on cells/genes critical for the development of humoral adaptive immune responses. Patterns of B cell and plasmacyte genes were altered in aging healthy gingival tissues. Substantial increases in a large number of genes reflecting antigen-dependent activation, B cell activation, B cell proliferation, and B cell differentiation/maturation were observed in periodontitis in adults and aged animals. Finally, evaluation of the relationship of these gene expression patterns with those of various tissue destructive molecules (MMP2, MMP9, CTSK, TNFα, and RANKL) showed a greater frequency of positive correlations in healthy tissues versus periodontitis tissues, with only MMP9 correlations similar between the two tissue types. These results are consistent with B cell response activities in healthy tissues potentially contributing to muting the effects of the tissue destructive biomolecules, whereas with periodontitis this relationship is adversely affected and enabling a progression of tissue destructive events

Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors

ABSTRACT In osteoclasts, Src controls podosome organization and bone degradation, which leads to an osteopetrotic phenotype in src ؊/؊ mice. Since this phenotype was even more severe in src ؊/؊ hck ؊/؊ mice, we examined the individual contribution of Hck in bone homeostasis. Compared to wt mice, hck ؊/؊ mice exhibited an osteopetrotic phenotype characterized by an increased density of trabecular bone and decreased bone degradation, although osteoclastogenesis was not impaired. Podosome organization and matrix degradation were found to be defective in hck ؊/؊ osteoclast precursors (preosteoclast) but were normal in mature hck ؊/؊ osteoclasts, probably through compensation by Src, which was specifically overexpressed in mature osteoclasts. As a consequence of podosome defects, the 3-dimensional migration of hck ؊/؊ preosteoclasts was strongly affected in vitro. In vivo, this translated by altered bone homing of preosteoclasts in hck ؊/؊ mice: in metatarsals of 1-wk-old mice, when bone formation strongly depends on the recruitment of these cells, reduced numbers of osteoclasts and abnormal developing trabecular bone were observed. This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling.-Vérollet, C., Gallois, A., Dacquin, R., Lastrucci, C., Pandruvada, S. M. N., Ortega, N., Poincloux, R., Behar, A., Cougoule, C., Lowell, C., Al Saati, T., Jurdic, P., Maridonneau-Parini, I. Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors. FASEB J. 27, 3608 -3618 (2013). www.fasebj.org Key Words: osteopetrosis ⅐ cell migration ⅐ podosomes ⅐ Src tyrosine kinases Bone is renewed continuously by a process known as bone remodeling. Bone remodeling is accomplished by 3 cell types: osteocytes, osteoblasts, and osteoclasts (OCs). Osteocytes are the mechanical sensors of bone that regulate osteoclast formation. Osteoblasts synthetize the matrix and promote its mineralization, while OCs are responsible for degradation of bones during bone development, homeostasis, and repair. The formation and degradation of bone are tightly balanced in both time and space. A dysregulation of this tight balance between bone formation and bone degradation may result either in loss of bone mass, such as in osteoporosis, or in contrast, in a progressive increase in bone mass, such as in osteopetrosis. Degrading OCs are large multinucleated giant cells formed by the differentiation and fusion of mononuclear monocyte lineage precursors after stimulation by receptor activator of nuclear factor -B ligand (RANKL) and macrophage colony-stimulationg factor (M-CSF) (1-3). They are characterized by high levels of cathepsin K and tartrate resistant acidic phosphatase (TRAP) activities, whic

Genome-wide haplotype-based association analysis of major depressive disorder in Generation Scotland and UK Biobank

Generation Scotland received core funding from the Chief Scientist Office of the Scottish Government Health Directorate CZD/16/6 and the Scottish Funding Council HR03006. Genotyping of the GS:SFHS samples was carried out by the Genetics Core Laboratory at the Wellcome Trust Clinical Research Facility, Edinburgh, Scotland and was funded by the Medical Research Council UK and the Wellcome Trust (Wellcome Trust Strategic Award “STratifying Resilience and Depression Longitudinally” (STRADL) Reference 104036/Z/14/Z. YZ acknowledges support from China Scholarship Council. IJD is supported by the Centre for Cognitive Ageing and Cognitive Epidemiology which is funded by the Medical Research Council and the Biotechnology and Biological Sciences Research Council (MR/K026992/1). AMMcI and T-KC acknowledges support from the Dr Mortimer and Theresa Sackler Foundation. We are grateful to all the families who took part, the general practitioners and the Scottish School of Primary Care for their help in recruiting them, and the whole Generation Scotland team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists, healthcare assistants and nurses. Ethics approval for the study was given by the NHS Tayside committee on research ethics (reference 05/S1401/8)Peer reviewedPublisher PD

Severe neurodegeneration with impaired autophagy mechanism triggered by Ostm1 deficiency

Loss of Ostm1 leads to the most severe form of osteopetrosis in mice and humans. Because functional rescue of the osteopetrotic defect in these mice extended their lifespan from ∼3 weeks to 6 weeks, this unraveled a second essential role of Ostm1. We discovered that Ostm1 is highly expressed in the mouse brain in neurons, microglia, and astrocytes. At 3–4 weeks of age, mice with Ostm1 loss showed 3–10-fold stimulation of reactive gliosis, with an increased astrocyte cell population and microglia activation. This inflammatory response was associated with marked retinal photoreceptor degeneration and massive neuronal loss in the brain. Intracellular characterization of neurons revealed abnormal storage of carbohydrates, lipids, and ubiquitinated proteins, combined with marked accumulation of autophagosomes that causes frequent axonal swelling. Stimulation of autophagy was provided by specific markers and by significant down-regulation of the mammalian target of rapamycin signaling, identifying a cellular pathologic mechanism. A series of transgenic mouse lines specifically targeted to distinct central nervous system cell subpopulations determined that Ostm1 has a primary and autonomous role in neuronal homeostasis. Complete functional complementation demonstrated that the development of severe and rapid neurodegeneration in these mice is independent of the hematopoietic lineage and has clinical implications for treatment of osteopetrosis. Importantly, this study establishes a novel neurodegenerative mouse model critical for understanding the multistep pathogenic cascade of cellular autophagy disorders toward therapeutic strategy design

In vitro evaluation of osteoblast responses to carbon nanotube-coated titanium surfaces

Abstract Background The effects of surface roughness and carboxyl functionalization of multi-walled carbon nanotubes (MWCNTs) mixed with collagen coated onto titanium (Ti) substrates on MC3T3-E1 osteoblasts were evaluated. Methods The proliferation, differentiation, and matrix mineralization were investigated using (1) smooth-surfaced Ti discs, (2) Ti discs coated with collagen and MWCNT (Ti-MWCNT), and (3) Ti discs coated with collagen and MWCNT-COOH (Ti-MWCNT-COOH) for applications in orthodontic mini screw implants (MSIs). The coatings were uniform when analyzed using scanning electron microscopy (SEM), and surface roughness was evaluated by surface profilometry that demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 μm) and MWCNT-COOH (0.84 ± 0.01 μm) groups. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay was performed after days 1, 3, and 7 to assess proliferation. Alkaline phosphatase (ALP)-specific activity was assessed after day 7 to quantify differentiation. Alizarin red staining was measured after day 28 to quantify matrix mineralization. All data were analyzed with JMP Pro11 software (SAS, USA) with a statistical significance of p  < 0.05. Results Surface profilometry demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 μm) and MWCNT-COOH (0.84 ± 0.01 μm) groups. On day 7, ALP assay showed that MWCNT-COOH (mean ± SD 0.98 ± 0.26 U/μg of protein) enhanced cell differentiation when compared to the uncoated group (p = 0.05). Alizarin red staining after 28 days of cell culture revealed that MWCNT-COOH (mean ± SD 1.5 ± 0.2 OD405) increased (p = 0.03) matrix mineralization when compared to the uncoated group (0.9 ± 0.09 OD405). Conclusions This study showed that coatings containing MWCNT-COOH (increased hydrophilic surface chemistry) influence osteoblast proliferation, differentiation, and matrix mineralization and should be further studied for applications in orthodontic MSIs

DAP12 overexpression induces osteopenia and impaired early hematopoiesis.

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis

Proliferation of spleen and bone marrow osteoclast precursors.

<p>Spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates at 78×10³ cells/well. At each time point, cells were incubated with BrdU during 3 hours. Incorporated BrdU was measured at 370 nm with a 492 nm reference wavelenght, using a colorimetric immunoassay. White circles: WT cells, black squares: Tg-hDAP12 cells. Results are means ± SE. <i>p</i><0,001 (***);<i>p</i><0,01 (**).</p

RANKL dose-dependent response of spleen and bone marrow osteoclast precursors.

<p>Bone marrow and spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates. 12,5×10³ cells/well for bone marrow cells and 78×10³per well for spleen cells. Bone marrow and spleen cells were grown in presence of M-CSF and increasing concentrations of RANKL either 20, 30, 50 and 100 ng/ml for bone marrow cells (A) or 20, 30, 40, 50 and 100 ng/ml for spleen cells (B). After 4 days in culture for bone marrow cells or 6 days in culture for spleen cells, TRAP-positive osteoclasts with ≥3 nuclei were counted. White bars:WT cells; Grey bars:Tg-hDAP12 cells. Results are expressed as mean number of osteoclasts with nuclei ≥3 present on the total surface of each of the three wells ± SE. In (B), ***indicated <i>p</i><0.001.</p

Osteoblastogenesis and osteoclastogenic activities of osteoblasts in bone marrow cell cultures from 3-month-old Tg-hDAP12 female mice.

<p>Osteoblasts were classically obtained after treatment of bone marrow cells with ß sodium-glycerophosphate (10 mM) and ascorbic acid (50 µg/ml) for 7 days. Total RNA was extracted at different times of culture: day 3, day 7 and day 14. (A) Gene expression of osteoblast-associated markers: alkaline phosphatase (ALP), osteocalcin (OCN), measured by RT-PCR at the indicated times of culture. Gene expression was normalized to the L32, house-keeping gene values. Results are means of two independent experiments ± SE. (B) Mineralized colonies obtained from osteoblasts generated from bone marrow. Colonies doubly-stained with alkaline phosphatase and von Kossa appear as black dots. Results are plotted as the mean number of nodules ±SE of three wells for 3-month-old WT and Tg-hDAP12 female mice and were representative of three independent experiments. (C) Expression of human DAP12 transgene in 7-day-old bone marrow-derived osteoblasts using RT-PCR. 1: WT osteoblasts; 2: Tg-hDAP12 osteoblasts. 3: hDAP12 expression in human monocyte-derived osteoclasts used as control; 4: RT-PCR without template. White stars indicate the 373 bp PCR fragment corresponding to hDAP12. One experiment is shown, representative of three independent experiments. Grey bars: Tg-hDAP12 mice; white bars: WT mice.</p