Toll-Like Receptor 2 Is Required for Inflammatory Process Development during Leishmania infantum Infection.

Visceral leishmaniasis (VL) is a chronic and fatal disease caused by Leishmania infantum in Brazil. Leukocyte recruitment to infected tissue is a crucial event for the control of infections such as VL. Among inflammatory cells, neutrophils are recruited to the site of Leishmania infection, and these cells may control parasite replication through oxidative or non-oxidative mechanisms. The recruitment, activation and functions of the neutrophils are coordinated by pro-inflammatory cytokines and chemokines during recognition of the parasite by pattern recognition receptors (PRRs). Here, we demonstrated that the Toll-like receptor 2 (TLR2) signaling pathway contributes to the development of the innate immune response during L. infantum infection. The protective mechanism is related to the appropriate recruitment of neutrophils to the inflammatory site. Neutrophil migration is coordinated by DCs that produce CXCL1 and provide a prototypal Th1 and Th17 environment when activated via TLR2. Furthermore, infected TLR2-/- mice failed to induce nitric oxide synthase (iNOS) expression in neutrophils but not in macrophages. In vitro, infected TLR2-/- neutrophils presented deficient iNOS expression, nitric oxide (NO) and TNF-α production, decreased expression of CD11b and reduced L. infantum uptake capacity. The non-responsive state of neutrophils is associated with increased amounts of IL-10. Taken together, these data clarify new mechanisms by which TLR2 functions in promoting the development of the adaptive immune response and effector mechanisms of neutrophils during L. infantum infection.

Skeletal actions of fasting-induced adipose factor (FIAF).

Several adipokines are known to influence skeletal metabolism. Fasting-induced adipose factor (FIAF) is an adipokine that gives rise to 2 further peptides in vivo, the N-terminal coiled-coil domain (FIAF(CCD)) and C-terminal fibrinogen-like domain (FIAF(FLD)). The skeletal action of these peptides is still uncertain. Our results show that FIAF(CCD) is a potent inhibitor of osteoclastogenesis and function, as seen in mouse bone marrow and RAW264.7 cell cultures, and in a resorption assay using isolated primary mature osteoclasts. The inhibitory effects at 500 ng/mL were approximately 90%, 50% and 90%, respectively, in these assays. FIAF(CCD) also stimulated osteoblast mitogenesis by approximately 30% at this concentration. In comparison, FIAF(FLD) was only active in decreasing osteoblast mitogenesis, and intact FIAF had no effect in any of these assays. In murine bone marrow cultures, FIAF(CCD) reduced the expression of macrophage colony-stimulating factor (M-CSF), nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP), and to lesser extent suppressed the expression of connective tissue growth factor (CTGF). FIAF(CCD) also decreased expression of M-CSF and CTGF in stromal/osteoblastic ST2 cells. Its effect on receptor activator of nuclear factor κB (RANKL) and osteoprotegerin expression in bone marrow was not consistent with its inhibitory action on osteoclastogenesis, but it decreased RANKL expression in ST2 cells. In RAW264.7 cell cultures, FIAF(CCD) significantly reduced the expression of NFATc1 and DC-STAMP. In conclusion, FIAF(CCD) inhibits osteoclast differentiation and function in vitro and decreases expression of genes encoding key osteoclastogenic factors such as M-CSF, CTGF, NFATc1, and DC-STAMP. FIAF(CCD)'s action on osteoclasts may be independent of the RANKL/osteoprotegerin pathway. These results suggest a novel mechanism by which adipose tissue may regulate bone resorption and skeletal health.

Extended treatment with selective phosphatidylinositol 3-kinase and mTOR inhibitors has effects on metabolism, growth, behaviour and bone strength.

The class I phosphatidylinositol 3-kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small-molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on-target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small-molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro-CT and three-point bending, respectively. Surprisingly, after sustained dosing with pan-PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug-naïve animals treated with the same drugs. However, pan-PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health.

Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro.

Ghrelin is released in response to fasting, such that circulating levels are highest immediately prior to meals. Bone turnover is acutely responsive to the fed state, with increased bone resorption during fasting and suppression during feeding. The current study investigated the hypothesis that ghrelin regulates the activity of bone cells. Ghrelin increased the bone-resorbing activity of rat osteoclasts, but did not alter osteoclast differentiation in a murine bone marrow assay nor bone resorption in ex vivo calvarial cultures. Ghrelin showed mitogenic activity in osteoblasts, with a strong effect in human cells and a weaker effect in rat osteoblasts. The expression of the human ghrelin receptor, GHSR, varied among individuals and was detectable in 25-30% of bone marrow and osteoblast samples. However, the rodent Ghsr expression was undetectable in bone cells and cell lines from rat and mouse. These data suggest that elevated levels of ghrelin may contribute to the higher levels of bone turnover that occurs in the fasted state.

Molecular mechanisms involved in the mitogenic effect of lactoferrin in osteoblasts.

Lactoferrin, an iron-binding glycoprotein present in milk and other exocrine secretions in mammals, is anabolic to bone at physiological concentrations. Lactoferrin stimulates the proliferation, differentiation and survival of osteoblasts, as well as potently inhibiting osteoclastogenesis in bone marrow cultures. In the current study we further investigated the mechanism of action of lactoferrin in osteoblasts. We used low-density arrays to measure the level of expression of 45 genes in MC3T3-E1 osteoblast-like cells treated with lactoferrin, and identified transient, dose-dependent increases in the transcription levels of interleukin-6, of the pro-inflammatory factor prostaglandin-endoperoxide synthase 2 (Ptgs2), and of the transcription factor nuclear factor of activated T cells (Nfatc1). We demonstrated similar changes in primary osteoblast cultures from human and rat. Levels of prostaglandin E2 were increased in conditioned media collected from osteoblasts treated with lactoferrin, indicating that the activity of the enzyme cyclooxygenase 2 (COX2), which is encoded by Ptgs2, was also up-regulated. Using a luciferase reporter construct we showed that lactoferrin induced transcription from the NFAT consensus sequence. We found that inhibiting either COX2 or NFATc1 activity blocked the mitogenic effect of lactoferrin in osteoblasts and that inhibition of NFATc1 activity partially blocked the transcriptional activation of Ptgs2. Our study has provided the first evidence that COX2 and NFATc1 activities are increased by lactoferrin, and demonstrated a role for each of these molecules as mediators of the mitogenic effects of lactoferrin in osteoblasts.

Skeletal phenotype of the leptin receptor-deficient db/db mouse.

Leptin, a major hormonal product of the adipocyte, regulates appetite and reproductive function through its hypothalamic receptors. The leptin receptor is present in osteoblasts and chondrocytes, and previously we have shown leptin to be an anabolic bone factor in vitro, stimulating osteoblast proliferation and inhibiting osteoclastogenesis. Leptin increases bone mass and reduces bone fragility when administered peripherally but also can indirectly reduce bone mass when administered into the central nervous system. However, data from animal models deficient in either leptin (ob/ob) or its receptor (db/db) remain contradictory. We compared the bone phenotype of leptin receptor-deficient (db/db) and wild-type mice using micro-computed tomographic (µCT) analysis of the proximal tibias and vertebrae. In the tibia, db/db mice had reduced percent trabecular bone volume (13.0 ± 1.62% in wild-type versus 6.01 ± 0.601% in db/db mice, p = .002) and cortical bone volume (411 ± 21.5 µm(3) versus 316 ± 3.53 µm(3), p = .0014), trabecular thickness (48.4 ± 001.07 µm versus 45.1 ± 0.929 µm, p = .041) and trabecular number (2.68 ± 0.319 mm(-1) versus 1.34 ± 0.148 mm(-1), p = .0034). In the fifth lumbar vertebral body, the trabecular thickness and cortical thickness were decreased in the db/db versus wild-type mice (0.053 ± 0.0011 mm versus 0.047 ± 0.0013 mm, p = .0002 and 0.062 ± 0.00054 mm versus 0.056 ± 0.0009 mm, p = .0001), respectively, whereas the trabecular and cortical percent bone volume and trabecular number did not reach significance. The total (endosteal and periosteal) cortical perimeter (12.2 ± 0.19 mm versus 13.2 ± 0.30 mm, p = .01) was increased. The serum osteocalcin levels were reduced in the db/db mice, suggesting that bone formation rates are decreased. The material properties of db/db femurs were determined by three-point bending and nanoindentation, showing decreased bone strength (13.3 ± 0.280 N versus 7.99 ± 0.984 N, p = .0074) and material stiffness (28.5 ± 0.280 GPa versus 25.8 ± 0.281 GPa, p < .0001). These results demonstrate that bone mass and strength are reduced in the absence of leptin signaling, indicating that leptin acts in vivo as an anabolic bone factor. This concurs with results of in vitro studies and of peripheral leptin administration in vivo and suggests that leptin's direct effects on bone cells are likely to override its actions via the central nervous system.

Genetic modifications of mouse proopiomelanocortin peptide processing.

Pro-opiomelanocortin (POMC) is a prohormone which undergoes extensive tissue and cell specific post-translational processing producing a number of active peptides with diverse biological roles ranging from control of adrenal function to pigmentation to the regulation of feeding. One approach to unraveling the complexities of the POMC system is to engineer mouse mutants which lack specific POMC peptides. We describe here the design, generation, validation, and preliminary analysis of one such partial POMC mutant specifically lacking α-MSH. In contrast to POMC null mutant mice, mice lacking α-MSH in the presence of all other POMC peptides maintain adrenal structures and produce corticosterone comparable to wildtype littermates; however, they still have decreased levels of aldosterone, as found in POMC null mutant mice. Our findings demonstrate that α-MSH is not needed for maintenance of adrenal structure or for corticosterone production, but is needed for aldosterone production. These data demonstrate that mouse strains generated with precise genetic modifications of POMC peptide processing can answer questions about POMC peptide function. Further analysis of this and additional strains of mice with modified POMC peptide processing patterns will open up a novel avenue for studying the roles of individual POMC peptides.

The atypical anti-psychotic clozapine decreases bone mass in rats in vivo.

BACKGROUND: Fracture risk is increased in patients with schizophrenia, who often receive long-term therapy with anti-psychotic drugs. The mechanisms by which skeletal fragility is increased in patients with psychosis include increased risk of falling, but direct skeletal toxicity of anti-psychotic drugs is a possibility that has not been investigated. METHODS: We examined the skeletal effects, in vivo and in vitro, of a typical anti-psychotic drug, haloperidol, which primarily inhibits dopaminergic signaling, and an atypical anti-psychotic drug, clozapine, which predominantly inhibits serotonergic signaling. RESULTS: In growing rats, 42 days of clozapine treatment reduced whole body bone mineral density by 15% (P<0.01 vs vehicle), and trabecular and cortical bone volume, as assessed by microcomputed tomography, by 29% and 15%, respectively (P<0.05 vs vehicle for each). Treatment with haloperidol did not affect bone density. Clozapine, but not haloperidol, transiently increased levels of serum corticosterone, and decreased levels of serum testosterone. In vitro, clozapine dose-dependently decreased osteoblast mitogenesis, osteoblast differentiation and osteoclastogenesis, while haloperidol did not affect any of these parameters. CONCLUSIONS: These data demonstrate that clozapine, but not haloperidol, exerts adverse skeletal effects in rodents, and that this effect may be attributable to direct actions to reduce osteoblast growth and function. Long-term administration of clozapine may therefore negatively affect bone health, and clinical studies to investigate this possibility are warranted.

In vitro and in vivo effects of adiponectin on bone.

Fat mass impacts on both bone turnover and bone density and is a critical risk factor for osteoporotic fractures. Adipocyte-derived hormones may contribute to this relationship, and adiponectin is a principal circulating adipokine. However, its effects on bone remain unclear. We have, therefore, investigated the direct effects of adiponectin on primary cultures of osteoblastic and osteoclastic cells in vitro and determined its integrated effects in vivo by characterizing the bone phenotype of adiponectin-deficient mice. Adiponectin was dose-dependently mitogenic to primary rat and human osteoblasts ( approximately 50% increase at 10 microg/ml) and markedly inhibited osteoclastogenesis at concentrations of 1 microg/ml or greater. It had no effect on osteoclastogenesis in RAW-264.7 cells or on bone resorption in isolated mature osteoclasts. In adiponectin knockout (AdKO) male C57BL/6J mice, trabecular bone volume and trabecular number (assessed by microcomputed tomography) were increased at 14 wk of age by 30% (P = 0.02) and 38% (P = 0.0009), respectively. Similar, nonsignificant trends were observed at 8 and 22 wk of age. Biomechanical testing showed lower bone fragility and reduced cortical hardness at 14 wk. We conclude that adiponectin stimulates osteoblast growth but inhibits osteoclastogenesis, probably via an effect on stromal cells. However, the AdKO mouse has increased bone mass, suggesting that adiponectin also has indirect effects on bone, possibly through modulating growth factor action or insulin sensitivity. Because adiponectin does influence bone mass in vivo, it is likely to be a contributor to the fat-bone relationship.

Plasma alpha-melanocyte-stimulating hormone: sex differences and correlations with obesity.

Rodent experiments raise the possibility of a regulatory role of peripheral alpha-melanocyte-stimulating hormone (alpha-MSH) in obesity and metabolism, but human data on peripheral alpha-MSH levels remain fragmentary. Because of the possible relationship between alpha-MSH and obesity, we endeavored to test the hypothesis that higher levels of alpha-MSH in obese patients would correlate with leptin levels and with other markers of obesity. Sixty normal-weight to obese healthy men and women participated. Weight, measures of body composition, and diet diaries were obtained; fasting blood was analyzed for alpha-MSH, lipids, glucose, insulin, leptin, and adiponectin. To begin to understand the source of peripherally measured hormones, alpha-MSH was also measured in serum samples from 5 individuals with untreated Addison disease. Levels of alpha-MSH were higher in men vs women (10.1 +/- 4.3 vs 7.6 +/- 3.4 pmol/L, P = .019), and alpha-MSH levels were higher in patients with Addison disease vs controls (17.7 +/- 2.3 vs 8.7 +/- 0.52 pmol/L, P < .001). Measures of adiposity correlated with insulin and leptin in men and women, and with adiponectin in women. alpha-Melanocyte-stimulating hormone levels did not correlate significantly with any parameter of adiposity or diet composition. The elevated alpha-MSH levels in patients with untreated Addison disease suggest possible pituitary secretion of alpha-MSH to the periphery. The lack of correlation between peripheral alpha-MSH and parameters of adiposity suggests that endogenous plasma alpha-MSH levels are not a metric for body composition per se.

Mutational analysis of evolutionarily conserved ACTH residues.

alpha-Melanocyte stimulating hormone (MSH) and adrenocorticotropin (ACTH)1-24, the minimal ACTH sequence required for full activity, differ only by the 10 C-terminal amino acids of ACTH1-24. Interestingly, these ten C-terminal residues have been highly conserved throughout vertebrate evolution. To understand the functional constraints of these 10 amino acids we analyzed the effects of mutating these residues on steroidogenic activity in vivo and in vitro. Alanine substitutions of some of the first four amino acid residues (the basic core residues KKRR, 15-18) greatly reduces ACTH activity in vitro and in vivo; replacement of mutant alanines at residues 15 and 17 with glutamine residues partially restores ACTH activity. Thus, for ACTH receptor binding and activation, the amino acid residues 15-18 are important for their side chains. Surprisingly, conversion of the five C-terminal residues (20-24) to alanines increases ACTH activity in vivo over that of native ACTH. With respect to receptor binding and activity, the last five amino acid residues are important only for the peptide length they contribute; however, with respect to serum stability, their side chains are significant.

Altered glucose homeostasis in proopiomelanocortin-null mouse mutants lacking central and peripheral melanocortin.

Prolonged obesity frequently leads to insulin resistance and, eventually, to diabetes. This relationship reflects the integration of fat stores and carbohydrate metabolism and the coordination of central nervous system functions, e.g. appetite, and peripheral metabolism. Recent work suggests that the melanocortin system is involved in this integration; specifically, central administration of melanocyte-stimulating hormone (MSH) decreases, whereas lack of central MSH signaling increases, peripheral insulin resistance. Here we asked whether MSH acting in the periphery has a complementary role in insulin resistance. We tested this in a mouse model where the proopiomelanocortin (POMC) gene encoding all of the melanocortins has been genetically deleted. The homozygous POMC-null mouse lacks central as well as peripheral MSH signaling; in addition, it lacks adrenal glands and thus is devoid of corticosterone and epinephrine. Here we report that homozygous POMC mutants have normal serum levels of insulin, normal fasting levels of glucose, and normal clearance of glucose in glucose tolerance tests. Thus, insulin production and sensitivity and glucose uptake in peripheral tissues are functioning normally. However, we found a striking inability of the homozygous POMC mutants to recover from insulin-induced hypoglycemia. This defect was in the glucagon-mediated counterregulatory response. Both peripheral administration of an MSH analog and supplementation with corticosterone alleviated the hypoglycemia after insulin challenge, but did not make the obese POMC mutant mice diabetic. We conclude that, similar to the regulation of body weight homeostasis, the regulation of glucose homeostasis requires the integration of both central and peripheral melanocortin signaling systems.