Severity Level and Duration of Energy Deficit in Mice Affect Bone Phenotype and Bone Marrow Stromal Cell Differentiation Capacity.

Anorexia nervosa is known to induce changes in bone parameters and an increase in bone marrow adiposity (BMA) that depend on the duration and seriousness of the disease. Previous studies have found that bone loss is associated with BMA accumulation. Sirtuin of type 1 (Sirt1), a histone deacetylase that is partly regulated by energy balance, was shown to have pro-osteoblastogenic and anti-adipogenic effects. To study the effects of the severity and duration of energy deficits related to bone loss, a mouse model of separation-based anorexia (SBA) was established. We recently demonstrated that moderate body weight loss (18%) 8-week SBA protocol in mice resulted in an increase in BMA, bone loss, and a significant reduction in Sirt1 expression in bone marrow stromal cells (BMSCs) extracted from SBA mice. We hypothesised that Sirt1 deficit in BMSCs is associated with bone and BMA alterations and could potentially depend on the severity of weight loss and the length of SBA protocol. We studied bone parameters, BMA, BMSC differentiation capacity, and Sirt1 expression after induction of 4 different levels of body weight loss (0%,12%,18%,24%), after 4 or 10 weeks of the SBA protocol. Our results demonstrated that 10 week SBA protocols associated with body weight loss (12%, 18%, 24%) induced a significant decrease in bone parameters without any increase in BMA. BMSCs extracted from 12% and 18% SBA groups showed a significant decrease in Sirt1 mRNA levels before and after co-differentiation. For these two groups, decrease in Sirt1 was associated with a significant increase in the mRNA level of adipogenic markers and a reduction of osteoblastogenesis. Inducing an 18% body weight loss, we tested a short SBA protocol (4-week). We demonstrated that a 4-week SBA protocol caused a significant decrease in Tb.Th only, without change in other bone parameters, BMA, Sirt1 expression, or differentiation capacity of BMSCs. In conclusion, this study showed, for the first time, that the duration and severity of energy deficits are critical for changes in bone parameters, BMSC differentiation, and Sirt1 expression. Furthermore, we showed that in this context, Sirt1 expression could impact BMSC differentiation with further effects on bone phenotype.

Cell-specific effects of TNF-α and IL-1ß on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification.

Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß stimulate tissue non-specific alkaline phosphatase (TNAP) activity and mineralization in cultures of vascular smooth muscle cells (VSMCs). They are, therefore, considered as stimulators of vascular calcification in the context of atherosclerosis and diabetes type 2. In contrast, although ankylosing spondylitis (AS) leads to the formation of syndesmophytes, which are ectopic ossifications from entheses (where ligaments, tendons and capsules are attached to bone), anti-TNF-α therapies fail to block bone formation in this disease. In this context, our aims were to compare the effects of TNF-α and IL-1ß on TNAP activity and mineralization in entheseal cells and VSMCs. Organotypic cultures of mouse ankle entheses were treated or not with TNF-α and IL-1ß for 5 days. Micro-computed tomography was performed to determine trabecular bone parameters, and histology to assess TNAP activity and mineralization. Human mesenchymal stem cells cultured in pellets in chondrogenic conditions and human VSMCs were also used to determine the effects of cytokines on TNAP activity and expression, measured by quantitative PCR. In organotypic cultures, TNF-α and IL-1ß significantly reduced the tibia BV/TV ratio. They also inhibited TNAP activity in entheseal chondrocytes in situ, and in mouse and human chondrocytes in vitro. In contrast, TNF-α stimulated TNAP expression and activity in human VSMCs. These differences were likely due to cell-specific effects of peroxisome proliferator-activated receptor γ (PPARγ), which is inhibited by TNF-α. Indeed, in human chondrocytes and VSMCs, the PPARγ inhibitor GW-9662 displayed the same opposite effects as TNF-α on TNAP expression. In conclusion, whereas TNF-α and IL-1ß stimulate TNAP activity in VSMCs, they inhibit it in entheseal cells in situ and on chondrocytes in vitro. The identification of PPARγ as a likely mediator of cytokine effects deserves consideration for future research on the mechanisms of ectopic ossification.

Sirtuin 1 deficiency decreases bone mass and increases bone marrow adiposity in a mouse model of chronic energy deficiency.

Sirtuin of type 1 (Sirt1), a class III HDAC, is known to be involved in the regulation of differentiation of skeletal stem cells (SSCs) into osteoblasts and adipocytes. In caloric restriction, it has been shown that the expression and activity of Sirt1 is a tissue-dependent regulation. However, at present, no study has focused on the link between Sirt1, bone marrow adiposity (BMA) and osteoporosis related to anorexia nervosa (AN). Thus, the aims of this work were to (i) determine BMA and bone changes in a mouse model replicating the phenotypes of AN (separation-based anorexia model (SBA)); (ii) determine the expression of Sirt1 in bone marrow stromal cells (BMSCs) extracted from these mice and identify their differentiation capacities; (iii) study the effects of pharmacological activation and inhibition of Sirt1 on the osteoblastogenesis and adipogenesis of these cells and (iiii) delineate the molecular mechanism by which Sirt1 could regulate osteogenesis in an SBA model. Our results demonstrated that SBA protocol induces an increase in BMA and alteration of bone architecture. In addition, BMSCs from restricted mice present a down-regulation of Sirt1 which is accompanied by an increase in adipogenesis at expense of osteogenesis. After a 10-day organotypic culture, tibias from SBA mice displayed low levels of Sirt1 mRNA which are restored by resveratrol treatment. Interestingly, this recovery of Sirt1 levels also returned the BMA, BV/TV and Tb.Th in cultured tibias from SBA mice to normal levels. In contrast of down-regulation of Sirt1 expression induced by sirtinol treatment, stimulation of Sirt1 expression by resveratrol lead to a decrease in adipogenesis and increase in osteogenesis. Finally, to investigate the molecular mechanisms by which Sirt1 could regulate osteogenesis in the SBA model, the acetylation levels of Runx2 and Foxo1 transcription factors were determined. Our data show that this chronic energy deficiency in female mice causes a decrease in BMSC activity, resulting in critical changes to Runx2 and Foxo1 acetylation levels and thus to their activity. Altogether, these data suggest that Sirt1 could be considered as a potential therapeutic target in osteoporosis related to AN.

Long-term physiological alterations and recovery in a mouse model of separation associated with time-restricted feeding: a tool to study anorexia nervosa related consequences.

BACKGROUND: Anorexia nervosa is a primary psychiatric disorder, with non-negligible rates of mortality and morbidity. Some of the related alterations could participate in a vicious cycle limiting the recovery. Animal models mimicking various physiological alterations related to anorexia nervosa are necessary to provide better strategies of treatment. AIM: To explore physiological alterations and recovery in a long-term mouse model mimicking numerous consequences of severe anorexia nervosa. METHODS: C57Bl/6 female mice were submitted to a separation-based anorexia protocol combining separation and time-restricted feeding for 10 weeks. Thereafter, mice were housed in standard conditions for 10 weeks. Body weight, food intake, body composition, plasma levels of leptin, adiponectin, IGF-1, blood levels of GH, reproductive function and glucose tolerance were followed. Gene expression of several markers of lipid and energy metabolism was assayed in adipose tissues. RESULTS: Mimicking what is observed in anorexia nervosa patients, and despite a food intake close to that of control mice, separation-based anorexia mice displayed marked alterations in body weight, fat mass, lean mass, bone mass acquisition, reproductive function, GH/IGF-1 axis, and leptinemia. mRNA levels of markers of lipogenesis, lipolysis, and the brown-like adipocyte lineage in subcutaneous adipose tissue were also changed. All these alterations were corrected during the recovery phase, except for the hypoleptinemia that persisted despite the full recovery of fat mass. CONCLUSION: This study strongly supports the separation-based anorexia protocol as a valuable model of long-term negative energy balance state that closely mimics various symptoms observed in anorexia nervosa, including metabolic adaptations. Interestingly, during a recovery phase, mice showed a high capacity to normalize these parameters with the exception of plasma leptin levels. It will be interesting therefore to explore further the central and peripheral effects of the uncorrected hypoleptinemia during recovery from separation-based anorexia.

A comparative study of antibodies against proteins extracted from staphylococcal biofilm for the diagnosis of orthopedic prosthesis-related infections in an animal model and in humans.

Staphylococcus aureus and Staphylococcus epidermidis are the microorganisms most frequently seen in periprosthetic infections (PPI) with the capacity of forming biofilm. To find potential antigens for the diagnosis of PPI, the immunogenicity of protein components in biofilm from a model biofilm-positive strain (S. epidermidis RP62A) was investigated. A guinea pig animal model of PPI was developed and sera were obtained. Sera of patients with PPI and those of controls were also collected. Data generated with an enzyme-linked immunosorbent assay showed that there were significantly higher levels of anti-extracellular protein IgG in sera of infected animals than in controls. We also found significantly higher anti-extracellular protein IgG levels in infected patients, compared to the controls; however, receiver operating characteristic curves did not aid in diagnosing PPI.

Neither the presence of ica locus, nor in vitro-biofilm formation ability is a crucial parameter for some Staphylococcus epidermidis strains to maintain an infection in a guinea pig tissue cage model.

The pathogenesis of Staphylococcus epidermidis is thought to be based on its capacity to colonize medical devices by forming a biofilm. Biofilm formation is in part mediated by the polysaccharide intercellular adhesin (PIA), which is encoded by the icaADBC operon. We have previously investigated in vitro the correlation existing between biofilm formation (B+/-), presence of ica locus (I+/-) and PIA production (P+/-) in some clinical isolates of coagulase-negative staphylococci (CoNS). Here, we used a guinea pig model of subcutaneous implanted tissue cages to assess the implication of B, I and P parameters in the capacity of nine S. epidermidis and one S. carnosus strains to develop and maintain an infection in vivo. Using clinical isolates and a model strain of S. epidermidis, we showed that the "B+, I+, P+" type confers the ability to maintain an infection in vivo. Surprisingly, the opposite type "B-, I-, P-" tested with clinical and commensal isolates, presented infection rates ranging from 25% to 60%. Other clinical isolates having a "B+, I+, P-" type, were not able to cause an infection in the present model. These results showed that, depending on the strains the capacity to colonize the tissue cage might be independent of the ability to form biofilm.

Potential use of poly-N-acetyl-beta-(1,6)-glucosamine as an antigen for diagnosis of staphylococcal orthopedic-prosthesis-related infections.

Staphylococcus aureus and coagulase-negative staphylococci are microorganisms most frequently isolated from orthopedic-implant-associated infections. Their capacity to maintain these infections is thought to be related to their ability to form adherent biofilms. Poly-N-acetyl-beta-(1,6)-glucosamine (PNAG) is an important constituent of the extracellular biofilm matrix of staphylococci. In the present study, we explored the possibility of using PNAG as an antigen for detecting antibodies in the blood sera of patients with staphylococcal orthopedic-prosthesis-associated infections. First, we tested the presence of anti-PNAG antibodies in an animal model, in the blood sera of guinea pigs that developed an implant-associated infection caused by biofilm-forming, PNAG-producing strains of Staphylococcus epidermidis. Animals infected with S. epidermidis RP62A showed levels of anti-PNAG immunoglobulin G (IgG) significantly higher than those of the control group. The comparative study of healthy individuals and patients with staphylococcal prosthesis-related infections showed that (i) relatively high levels of anti-PNAG IgG were present in the blood sera of the healthy control group, (ii) the corresponding levels in the infected patients were slightly but not significantly higher, and (iii) only 1 of 10 patients had a level of anti-PNAG IgM significantly higher than that of the control group. In conclusion, the encouraging results obtained in the animal study could not be readily applied for the diagnosis of staphylococcal orthopedic-prosthesis-related infections in humans, and PNAG does not seem to be an appropriate antigen for this purpose. Further studies are necessary to determine whether the developed enzyme-linked immunosorbent assay method could serve as a complementary test in the individual follow-up treatment of such infections caused by PNAG-producing staphylococci.