Fiona Collier

Selective serotonin reuptake inhibitors inhibit human osteoclast and osteoblast formation and function.

BACKGROUND Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants and one of the most commonly used medications. There is growing concern that SSRIs, which sequester in bone marrow at higher concentrations than brain or blood, increase bone fragility and fracture risk. However, their mechanism of action on human osteoclasts (OC) and osteoblasts (OB) differentiation remains unclear. METHODS Expression of serotonin receptors (5-HTR), transporter (5-HTT), and tryptophan hydroxylase 1 (TPH1) was assessed in human OC (precursors and mature) and OB (nonmineralizing and mineralizing) by polymerase chain reaction. OC formation and resorption was measured in the presence of 5 SSRIs. OBs cultured with SSRIs for 28 days were assessed for alkaline phosphatase (ALP) and bone mineralization. Cell viability and apoptosis were determined by annexin V flow cytometry. RESULTS OCs and OB expressed TPH1, 5-HTT, and 5-HTR1B. The 5-HTR2A was expressed only in OB, whereas 5-HTR2B expression increased from precursor to mature OC. All SSRIs (except citalopram) dose-dependently inhibited OC formation and resorption between 1 μmol/L and 10 μmol/L; order of potency: sertraline > fluoxetine > paroxetine > fluvoxamine > citalopram. Similarly, SSRIs (except citalopram) inhibited ALP and bone mineralization by OB but only at 30 μmol/L. Apoptosis was induced by SSRIs in OC and OB in an identical pattern to inhibitory effects. Serotonin treatment had no effect on either OC or OB parameters. CONCLUSIONS These data demonstrate that SSRIs differentially inhibit bone cell function via apoptosis. This may explain the mechanisms of bone loss with chronic use and aid clinical choices.

M-CSF Potently Augments RANKL-Induced Resorption Activation in Mature Human Osteoclasts

Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit-granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFκB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200–300%) stimulation at 25 ng/mL, an effect observed within 4–6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFκB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos.

Cord Blood-Derived Macrophage-Lineage Cells Rapidly Stimulate Osteoblastic Maturation in Mesenchymal Stem Cells in a Glycoprotein-130 Dependent Manner

In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC) generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors) induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate) these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF) to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL) to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM) greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM), and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2) actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation) or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells, including osteoclasts but not classically activated macrophages, can strongly drive MSC-osteoblastic commitment in OSM-dependent manner. This supports the notion that eliciting gp130-dependent signals in human MSC would be a useful approach to increase bone formation.

Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion

Background: Skeletal muscle fiber formation requires myoblast cell-cell membrane contact and fusion. Results: A versican-rich pericellular matrix surrounding myoblasts is proteolytically cleared by ADAMTS versicanases facilitating myoblast contact and fusion. Conclusion: Versican processing by ADAMTS versicanases contribute to muscle fiber formation. Significance: Targeting versican remodeling could enhance the regenerative capacity of muscle by improving muscle fiber fusion during regeneration. Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

Cord blood monocyte-derived inflammatory cytokines suppress IL-2 and induce nonclassic "T(H)2-type" immunity associated with development of food allergy

Infants who develop food allergy display hyperresponsive innate immunity at birth that promotes nonclassical TH2 differentiation. Fighting food allergy For people with food allergies, a slice of pizza or a peanut butter sandwich can be deadly. Yet despite the increasing prevalence of food allergy, little is known as to the immunological causes. Now, Zhang et al. report that infants who later developed food allergy had altered immunity at birth. Cord blood from these infants had more monocytes compared with CD4+ T cells and decreased numbers of regulatory T cells. Moreover, the monocytes from food-allergic infants secreted more inflammatory cytokines than those from healthy infants. These cytokines suppressed interleukin-2 (IL-2) expression by CD4+ T cells and skewed differentiation of these cells to a nonclassical T helper 2 (TH2) phenotype. Anti-inflammatory strategies should therefore be considered in preventing food allergy in these individuals. Food allergy is a major health burden in early childhood. Infants who develop food allergy display a proinflammatory immune profile in cord blood, but how this is related to interleukin-4 (IL-4)/T helper 2 (TH2)–type immunity characteristic of allergy is unknown. In a general population-derived birth cohort, we found that in infants who developed food allergy, cord blood displayed a higher monocyte to CD4+ T cell ratio and a lower proportion of natural regulatory T cell (nTreg) in relation to duration of labor. CD14+ monocytes of food-allergic infants secreted higher amounts of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor–α) in response to lipopolysaccharide. In the presence of the mucosal cytokine transforming growth factor–β, these inflammatory cytokines suppressed IL-2 expression by CD4+ T cells. In the absence of IL-2, inflammatory cytokines decreased the number of activated nTreg and diverted the differentiation of both nTreg and naïve CD4+ T cells toward an IL-4–expressing nonclassical TH2 phenotype. These findings provide a mechanistic explanation for susceptibility to food allergy in infants and suggest anti-inflammatory approaches to its prevention.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line.

Lineage‐specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell‐free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell‐free matrices from a murine bone marrow stromal cell line (MS‐5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellular matrices were used as biological scaffolds for the lineage‐specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony‐forming assays. We could get up to 80‐fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering‐based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow. Copyright

Cohort Profile: The Barwon Infant Study

The modern environment is associated with an increasing burden of non-communicable diseases (NCDs). Mounting evidence implicates environmental exposures, experienced early in life (including in utero), in the aetiology of many NCDs, though the cellular/molecular mechanism(s) underlying this elevated risk across the life course remain unclear. Epigenetic variation has emerged as a candidate mediator of such effects. The Barwon Infant Study (BIS) is a population-derived birth cohort study (n = 1074 infants) with antenatal recruitment, conducted in the south-east of Australia (Victoria). BIS has been designed to facilitate a detailed mechanistic investigation of development within an epidemiological framework. The broad objectives are to investigate the role of specific environmental factors, gut microbiota and epigenetic variation in early-life development, and subsequent immune, allergic, cardiovascular, respiratory and neurodevelopmental outcomes. Participants have been reviewed at birth and at 1, 6, 9 and 12 months, with 2- and 4-year reviews under way. Biological samples and measures include: maternal blood, faeces and urine during pregnancy; infant urine, faeces and blood at regular intervals during the first 4 years; lung function at 1 month and 4 years; cardiovascular assessment at 1 month and 4 years; skin-prick allergy testing and food challenge at 1 year; and neurodevelopmental assessment at 9 months, 2 and 4 years. Data access enquiries can be made at [www.barwoninfantstudy.org.au] or via [peter.vuillermin@deakin.edu.au].

The effects of maternal anxiety during pregnancy on IGF2/H19 methylation in cord blood

Compelling evidence suggests that maternal mental health in pregnancy can influence fetal development. The imprinted genes, insulin-like growth factor 2 (IGF2) and H19, are involved in fetal growth and each is regulated by DNA methylation. This study aimed to determine the association between maternal mental well-being during pregnancy and differentially methylated regions (DMRs) of IGF2 (DMR0) and the IGF2/H19 imprinting control region (ICR) in newborn offspring. Maternal depression, anxiety and perceived stress were assessed at 28 weeks of pregnancy in the Barwon Infant Study (n=576). DNA methylation was measured in purified cord blood mononuclear cells using the Sequenom MassArray Platform. Maternal anxiety was associated with a decrease in average ICR methylation (Δ=−2.23%; 95% CI=−3.68 to −0.77%), and across all six of the individual CpG units in anxious compared with non-anxious groups. Birth weight and sex modified the association between prenatal anxiety and infant methylation. When stratified into lower (⩽3530 g) and higher (>3530 g) birth weight groups using the median birth weight, there was a stronger association between anxiety and ICR methylation in the lower birth weight group (Δ=−3.89%; 95% CI=−6.06 to −1.72%), with no association in the higher birth weight group. When stratified by infant sex, there was a stronger association in female infants (Δ=−3.70%; 95% CI=−5.90 to −1.51%) and no association in males. All the linear regression models were adjusted for maternal age, smoking and folate intake. These findings show that maternal anxiety in pregnancy is associated with decreased IGF2/H19 ICR DNA methylation in progeny at birth, particularly in female, low birth weight neonates. ICR methylation may help link poor maternal mental health and adverse birth outcomes, but further investigation is needed.

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

The Potential Link between Gut Microbiota and IgE-Mediated Food Allergy in Early Life

There has been a dramatic rise in the prevalence of IgE-mediated food allergy over recent decades, particularly among infants and young children. The cause of this increase is unknown but one putative factor is a change in the composition, richness and balance of the microbiota that colonize the human gut during early infancy. The coevolution of the human gastrointestinal tract and commensal microbiota has resulted in a symbiotic relationship in which gut microbiota play a vital role in early life immune development and function, as well as maintenance of gut wall epithelial integrity. Since IgE mediated food allergy is associated with immune dysregulation and impaired gut epithelial integrity there is substantial interest in the potential link between gut microbiota and food allergy. Although the exact link between gut microbiota and food allergy is yet to be established in humans, recent experimental evidence suggests that specific patterns of gut microbiota colonization may influence the risk and manifestations of food allergy. An understanding of the relationship between gut microbiota and food allergy has the potential to inform both the prevention and treatment of food allergy. In this paper we review the theory and evidence linking gut microbiota and IgE-mediated food allergy in early life. We then consider the implications and challenges for future research, including the techniques of measuring and analyzing gut microbiota, and the types of studies required to advance knowledge in the field.