Erica Ramassa
Elena Sapei
Introduction
Osteoporosis is a common disease in postmenopausal women and older people associated with progressive loss of bone mineral density (BMD) and compromised bone strength, with increasing risk of fracture over time. Investigational therapies to reduce the loss of BMD include anti-inflammatory dietary supplementations, such as polyunsaturated fatty acids (PUFAs).
Although current data are insufficient, it has been suggested that PUFAs and saturated fatty acids can influence bone growth and modeling in humans.
The modern diet, with higher intake of fat has resulted in a higher ratio of omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acids, contributing to low-grade chronic inflammation (LGCI) and thus promoting the development of many chronic diseases, including osteoporosis.
There is evidence that overconsumption of n-6 PUFA coupled with under-consumption of n-3 PUFA results in LGCI and, with the increased presence of reactive oxygen species, leads to a shift in mesenchymal stem cells (precursors for both osteoblasts and adipocytes) lineage commitment toward increased adipogenesis and suppressed osteoblastogenesis.
According to numerous anthropological and epidemiological studies, humans evolved on a diet with a n-6 to n-3 PUFA ratio of approximately 1-2:1, whereas this ratio in the current Western-type diet has increased to approximately 10-15:1.
Low-grade chronic inflammation perpetuated by modern diet as a promoter of obesity and osteoporosis., 2014
The role of the Immune system in the osteoporosis
Proinflammatory cytokines, such as IL-1, IL-6 and TNF-alpha, promotes osteoclastogenesis and suppress osteoblastogenesis. Moreover, activated B and T cells can be potent regulators of bone resorption. Activated B cells can produce RANKL ,the activator ligand of osteoclasts’ receptor RANK, as well as other cytokines that are involved in bone resorption and bone formation. In addition, a subpopulation of memory B cells also expresses RANKL.
Estrogens are potent regulators of B-lymphopoiesis at a very early stage. In mice the absolute number of B cells (defined by expression of the antigen CD45R/B200) in bone marrow roughly doubles after ovariectomy. Because B cells at multiple stages of differentiation can express RANKL, it is possible that changes in early B cells with estrogen withdrawal may affect osteoclastogenesis and subsequent bone loss. However, this response does not appear involved in the bone loss that occurs after estrogen withdrawal in humans because increases in B cells were not found in postmenopausal women who had their estrogen replacement stopped.
How B Cells Influence Bone Biology in Health and Disease., 2010
Estrogen deficiency, typical of menopausal status, induces bone loss, up-regulates TNF-alpha production by T cells and increases the expression of RANKL on lymphocytes and marrow stromal cells. Ovariectomy stimulates the expression of CIITA and its product, a transcriptional co-activator when recruited to the MHCII promoter, stimulates antigen presentation in bone marrow monocytes with the final effect of up-regulation of expression of MHCII on macrophages. So, in the bone marrow, ovariectomy promotes T cell activation, by increasing antigen presentation by macrophages and DCs, and increases the number of bone marrow T cell-producing TNF.
The co-stimulation by RANKL and M-CSF is essential for the differentiation of monocytes into osteoclasts. The Receptor Activator of NFkB Ligand (RANKL) and Macrophage Colony Stimulating Factor (M-CSF) are cytokines produced by bone marrow stromal cells, osteoblasts and activated T cells. M-CSF induces the proliferation of osteoclasts’ precursors, differentiation and fusion of more mature osteoclasts and increases the survival of mature osteoclasts. RANKL, a member of the TNF superfamily, promotes the differentiation of osteoclast precursors into fully mature multinucleated osteoclasts and stimulates the capacity of mature osteoclasts to resorb bone.
The role of the immune system in the physiopathology of osteoporosis, 2012
How B Cells Influence Bone Biology in Health and Disease., 2010
TNFalpha, overproduced by T cells in estrogen deficiency, enhances osteoclasts’ formation by up-regulating stromal cell production of RANKL and M-CSF and by increasing the responsiveness of osteoclasts’ precursors to RANKL. IL-1 promotes RANKL expression by bone marrow stromal cells and osteoblasts and stimulates osteoclasts’ precursor and osteoclasts’ differentiation, lifespan and activity. TNF and IL-1 engage initially distinct signaling pathway that converge with the activation of the transcription factor NF-kB and the stimulation of the miogeno-activated protein kinase (MAPK) system. Thus, the combined effect of these two cytokines provides a potent signal to osteoclastogenesis, inhibition of osteoblasts’ function, and regulation of the lifespan of skeletal cells. IFNgamma influences osteoclasts’ formation both via direct inhibitory and indirect activatory effects. It directly blocks osteoclasts’ formation targeting maturing osteoclasts and also induces antigen presentation and thus T cell activation. When IFNgamma levels are increased in vivo, activated T cells secrete pro-osteoclastogenic factors and this activity off-sets the anti-osteoclastogenic effect of IFNgamma.
An inflammatory milieu induces naive T cells to differentiate into Th17, capable to produce RANKL, TNFalpha and IL-17, a cytokine that increases RANKL expression by osteoblasts, and induces, as suggested by some data, dendritic cells (DC) to act as osteoclasts’ precursors, transforming into DC-derived-osteoclasts according to phenotypic and functional characterization studies.
The role of the immune system in the physiopathology of osteoporosis, 2012
Studies have demonstrated that also IL-7 plays an important role in the regulation of bone homeostasis. However, the precise nature of how IL-7 affects osteoclasts and osteoblasts is controversial. Systemic administration of IL-7 increased osteoclast formation from human peripheral blood cells by increasing osteoclastogenic cytokine production in T cells. Furthermore, mice with global over expression of IL-7 had a phenotype of decreased bone mass with increased osteoclasts and no change in osteoblasts. In addition, IL-7 has secondary effects, which result from the production of bone-resorbing cytokines by T cells in response to activation by this cytokine. However, according to other studies, IL-7 inhibited osteoclast formation in murine bone marrow cells that were cultured for 5 days with M-CSF and RANKL and IL-7- deficient mice had markedly increased osteoclast number and decreased trabecular bone mass compared to wild-type controls.
How B Cells Influence Bone Biology in Health and Disease., 2010
B cells have recently been directly implicated in the regulation of bone resorption as they represent a major source of Osteoprotegerin (OPG), an inhibitor of RANK. In fact, B cell Knockout (KO) mice present at baseline a specific deficiency in OPG (mRNA and protein) and an osteoporotic phenotype, a consequence of enhanced osteoclastic bone resorption. Reconstitution of young B cell KO mice with B cell by means of adoptive transfer, completely rescued mice from development of osteoporosis, by normalizing OPG production. B cells also play an important role in regulating basal osteoclasts’ formation and in regulating bone omeostasis.
Data suggest that B-lymphocyte involvement in the adaptive immune response contributes to bone resorption by up-regulating of RANKL expression through Toll-like receptor pathways. These data align with the known ability of T cells to produce RANKL in the presence of immune stimulus and to increase osteoclastogenesis.
The role of the immune system in the physiopathology of osteoporosis, 2012
In bone marrow a series of interactions occur, some of which are mediated by B cell Transcriptional Factors (TFs) that regulate the balance between B cell and macrophage differentiation from progenitors. Loss of these key regulatory TFs (e.g. Ikaros, Ebf1 and Pax5) results in lineage allocation being shifted away from B cell differentiation and towards myeloid lineage differentiation.
IL-7 is the major growth factor for B cells and is expressed by bone marrow stromal cells and osteoblasts and PU.1 is the Transcription Factor that regulates the IL-7Rs. High levels of PU.1 expression promotes macrophage differentiation and blocks B cell development, while low concentrations of PU.1 protein promote B cell differentiation. Thus, PU.1 is required for the generation of lymphoid progenitors but not for their further differentiation. That is the responsibility of E2A, Ebf1 and Pax5. In the myeloid lineage, PU.1 is required for the generation of myeloid progenitors and the differentiation of macrophages and osteoclasts.
Although osteoclasts and B cells arise from different progenitors there are data to support the hypothesis that B cells and macrophages can differentiate from a common progenitor downstream from the multipotential progenitor cells (MPP). A rare population (0.5%) of B cell progenitors was isolated from adult mouse BM cells and was CD45R/B220 CD19 . When cultured in myeloid stimulating conditions these cells gave rise to macrophages indicating the bi-potential nature of these cells. The loss of Pax5 causes the expression of unwanted genes, including c-fms, the M-CSF receptor that supports macrophage and osteoclasts’ differentiation. Therefore, loss of Pax5 could support the bi-potential progenitor population.
Moreover, loss of members of the RANK, RANKL, OPG pathway not only affects osteoclasts differentiation but also B cell development. In fact, the loss of OPG increases pro-and mature B cells, while the loss of RANKL or RANK decreases these populations of cells.
How B Cells Influence Bone Biology in Health and Disease., 2010
Polyunsatured fatty acids (PUFAs)
According to their structure, these fatty acids are divided into two categories depending on the saturation condition of the molecule. The terms n-3 and n-6 denote that the third and sixth carbon, respectively, from the methyl terminal is unsaturated. The n-3 and n-6 fatty acids can originate from the dietary 18-carbon precursors alpha-linolenic acid and linoleic acid, respectively. Dietary sources of long-chain n-3 and n-6 fatty acids are essential because mammals cannot synthesize such fatty acids. In addition, mammalian cells cannot convert n-6 to n-3 fatty acids because they lack the converting enzyme named n-3 desaturase. Dietary intake of essential fatty acids has a crucial role in the cell membrane and the production of various cytokines by inflammatory cells. The n-6 fatty acids, especially arachidonic acid (AA), are the primary source of the n-6 eicosanoids that are produced from the oxygenation of AA by cyclooxygenase, lipoxygenase, and epoxygenase enzymes to produce prostaglandins, leukotrienes, lipoxines and P-450 compounds. After the digestion of fish or fish oil, the dietary long-chain n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) partially replace the n-6 fatty acids, particularly AA, in the membranes of platelets, erythrocytes, neutrophils, monocytes, and liver cells.
A cura del Dr. Gianluca Rizzo,www.my-personaltrainer.it
An higher introduction of n-3 fatty acids with the diet can change the ratio of n-6/n-3 fatty acids in membranes and a change in their function can decrease the production of IL-1, IL-6 and TNF-a.
Eicosanoids, the metabolic products of the sequential elongation and desaturation processes of both n-6 and n-3 PUFA, are signaling molecules responsible for the immune and inflammatory reactions in the body. The major metabolites of n-6 PUFA are proinflammatory eicosanoids and include prostaglandin E2, leukotriene B4, and thromboxane A2, generated by lipid-oxidizing enzyme lipoxygenase-5 and cyclooxygenase-2. The major metabolites of n-3 PUFA are the anti-inflammatory eicosanoids and include prostaglandin E3, leukotriene B5, resolvins, and protectins, maresins also generated by COX-2 and 5-LOX. During metabolism, n-6 and n-3 PUFAs compete for the desaturation enzymes and the higher amount of n-6 PUFA in the modern diet shifts the balance toward the production of proinflammatory eicosanoids.
Acute inflammation activates prostaglandins, leukotrienes, and inflammatory cytokines such as TNF-a, IL-6 and IL-1 which recruit neutrophils and macrophages. When the insult is dealt with resolvins, protectins, maresins stop further recruitment of immune cells and the propagation of pro-inflammatory cytokines, so that the cell can return to its basal state.
During LGCI (Low Grade Chronic Inflammation) inflammation is not resolved; this state results from the continuous presence of specific immune cells leading to 2-4-fold elevations in the circulating levels of pro- and anti-inflammatory cytokines, and more are recruited in a perpetual cycle. The exact PUFA metabolite composition of this unresolved or surplus inflammation is unknown, but it is likely that there is a shift towards the proinflammatory eicosanoids, particularly with higher n-6 PUFAs intake.
Low-grade chronic inflammation perpetuated by modern diet as a promoter of obesity and osteoporosis. 2014
Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS, Arh Hig Rada Toksikol 2014;65:139-148
The mechanisms of action of dietary fatty acids on bone
A wide range of mechanisms may mediate the effects of dietary fats on bone, including alterations in calcium absorption and urinary calcium loss, prostaglandin synthesis, osteoclast formation, and lipid oxidation.
Adult bone is continually renewed by a coupled process of formation and resorption. Bone reorganization starts with osteoclasts, which are a subpopulation of hematopoietic cells.
Osteoblasts and osteoclasts interact; the receptor activator of nuclear factor-kB ligand (RANKL) is expressed by osteoblasts and activates its receptor, RANK, which is expressed on osteoclasts, thus promoting osteoclast formation and activation, as well as suppressing apoptosis of osteoclasts. Osteoprotegerin (OPG) is a secretory glycoprotein expressed by osteoblasts which blocks RANKL from activating RANK. The ratio of OPG/RANKL is critical in the pathogenesis of resorptive bone disease, with a higher ratio indicating less bone resorption.
The effect of n-3 PUFAs on nuclear factor-kappaB (NF-κB) has been examined in vitro. Pre-treatment of osteoclasts with EPA decreased tumor necrosis factor α (TNF α)-induced NF-κB protein expression and activation in a dose-dependent manner. The n-3 PUFAs have been shown to decrease production of the n-6 PUFA derived eicosanoid, prostaglandin E2 (PGE2), and increase bone formation markers, alkaline phosphatase and osteocalcin. High PGE2 decreases OPG production and increases RANKL expression. DHA added to osteoblastic cell cultures does not stimulate RANKL, but the n-6 PUFA, arachidonic acid, stimulates RANKL and inhibits OPG secretion by 25–30% thus reducing the OPG/RANKL ratio.
A systematic review of omega-3 fatty acids and osteoporosis. 2012
It was demonstrated that DHA, not EPA, inhibited osteoclastogenesis induced by the receptor activator of nuclear factor-κB ligand (sRANKL) in vitro. In the study of Akiyama M. et al, it was performed gene expression analysis using microarrays to identify genes affected by the DHA treatment during osteoclastogenesis. Quantitative PCR analysis confirmed that DC-STAMP, an essential gene in osteoclastogenesis, and other osteoclast-related genes, such as Siglec-15, Tspan7 and Mst1r, were inhibited by DHA. These findings may contribute to the molecular understanding of the beneficial effects of DHA as a food supplement.
Impact of docosahexaenoic acid on gene expression during osteoclastogenesis in vitro--a comprehensive analysis. 2013
A second possible mechanism by which n-3 PUFAs may influence bone is related to up-regulation of intestinal calcium absorption. Essential PUFAs are necessary for maximal vitamin D-dependent calcium absorption. In vitro and animal studies show up-regulation of duodenal calcium absorption by DHA via modulating Calcium-ATPase when calcium levels are low. In ovariectomized rats, calcium-ATPase activity at the intestinal basolateral membrane increases significantly following supplementation with essential PUFAs in a ratio of 1/3, GLA/EPA + DHA. DHA and EPA decrease urinary calcium excretion in a rat model of osteoporosis exacerbated by restricted food intake and in patients with idiopathic calcium nephrolithiasis, fish oil decreases urinary calcium excretion and returned the high level of calcium absorption toward normal. Thus, the essential n-3 and n-6 PUFAs may act to increase calcium absorption and decrease calcium excretion, especially when dietary calcium intake is low.
A systematic review of omega-3 fatty acids and osteoporosis. 2012
B. Y. Y. Lau et al, Investigating the Role of Polyunsaturated Fatty Acids in Bone Development Using Animal Models, Molecules 2013, 18, 14203-14227 Investigating the role of polyunsaturated fatty acids in bone development using animal models. 2013
Moreover, it has been established that reactive oxygen species (ROS) and oxidative stress are one of the key factors in the regulation of bone metabolism. Osteoporotic patients have significantly increased oxidative stress compared with healthy controls and that examination of isolated osteoclasts in vitro has indicated that mature osteoclasts generate ROS.
Three inflammatory cytokines, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-a), have been shown to be active in the pathogenesis of osteoporosis. IL-1 and TNF-a are potent stimulators of bone resorption and inhibitors of bone formation and also stimulate IL-6 production. IL-6 stimulates osteoclastogenesis in the bone and, simultaneously, higher amount of fat mass activates osteoclasts via increase in IL-1 and TNF-a. Osteoclasts are derived from the same hematopoietic stem cells as the immune cells. In Low Grade Chronic Iinflammation (LGCI), osteoclastogenesis and bone resorption are accelerated, as the osteoclasts are programmed to respond to inflammatory signals.
A systematic review of the impact of n-3 fatty acids in bone health and osteoporosis. 2008
Mesenchymal stem cells (MSC) are the common precursors for osteoblasts and adipocytes. Even under normal homeostatic conditions, MSC slightly favors adipogenic differentiation. In LGCI, pro-inflammatory mediators in the presence of a high n-6 to n-3 ratio and reactive oxygen species (ROS), stimulate adipogenesis and suppress osteoblastogenesis, ensuring that MSC are committed to becoming adipocytes.
In addiction, the enzyme COX-2 has a higher affinity for n-6 than n-3 PUFAs and n-6 will be metabolized at a higher rate. However, higher presence of n-3, especially at the 1:1 ratio, would increase the amount of n-3 metabolized, which would lower the production of inflammatory eicosanoids.
Low-grade chronic inflammation perpetuated by modern diet as a promoter of obesity and osteoporosis. 2014
Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS, Arh Hig Rada Toksikol 2014;65:139-148 Low-grade chronic inflammation perpetuated by modern diet as a promoter of obesity and osteoporosis. 2014
In osteoporotic bone, reduced bone formation is directly correlated with increasing adipose tissue. PPARγ and RUNX2 are key transcriptional factors that regulate respectively adipogenesis and osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs). In the study of Tian-yu Chen et al, for better understanding the effect of endogenous n-3 PUFAs, TG mice with FAT 1 gene overexpression were developed, which was able, by converting n-6 PUFAs to n-3 PUFAs endogenously, to show a much lower n-6:n-3 PUFA ratio (<1:1) compared to WT mice with ratio of 20–30:1. It was observed a significantly lower expression of PPARγ but higher RUNX2 expression in bone marrow of TG mice when compared to WT mice, which confirmed the inhibitory effect of n-3 PUFAs on bone marrow adipogenesis theoretically. In addition, after ovariectomy, situation that induced bone loss, TG mice presented significantly lower bone marrow adiposity (adipocyte volume/tissue volume, mean adipocyte number) than WT mice, but increased the bone parameters (bone mineral density, bone mineral content, bone volume/total volume) in the distal femoral metaphysis.
Endogenous n-3 polyunsaturated fatty acids (PUFAs) mitigate ovariectomy-induced bone loss by attenuating bone marrow adipogenesis in FAT1 transgenic mice. 2013
Results of studies on humans
Some epidemiological evidence suggests that diets high in omega 3 fatty acids (n-3 PUFAs) may be beneficial for skeletal health. But, though the association of n-3 PUFAs to bone turnover markers appears promising, the real test of clinical significance is the impact of n-3 PUFAs on osteoporotic fracture and epidemiological studies, that, in a small number, investigated the relationship of fish consumption or dietary n-3 PUFA consumption to fracture risk, have yielded mixed results.
A systematic review, with the aim of determining if randomized controlled trials (RCTs) support a positive effect of n-3 PUFAs on osteoporosis, included 10 RCTs. This review highlights three problems related to the way in which the RCTs are conducted:
- Race/ethnicity impacts BMD and is a known risk factor for fractures (for example, white women have about a 60% higher risk for hip fractures than black women and about a 75% higher risk than Asian/Pacific Island Women). One of the limitations of most of the reviewed RCTs is the failure to report the racial/ethnic background of the participants. So, future studies investigating n-3 PUFAs and skeletal health need to address the paucity of data in multi-ethnic cohorts.
- The source of n-3 PUFAs may also have a significant impact on skeletal health. In fact, different mixtures of n-3 (either alone or in combination for example with calcium and / or vitamins) or n-3 from different sources have different effects.
- It is also important to consider the oils or foods used as placebos for n-3 PUFA trials. Saturated, monounsaturated and n-6 polyunsaturated fatty acids found in various ratios in olive oil, corn oil, coconut oil and wheat germ (all placebos used in RCTs in this review) may impact inflammatory pathways, calcium absorption, bone turnover and BMD differentially, making the detection of an effect from n-3 PUFA intervention more or less likely depending on the choice of placebo.
The conclusions of the review are: four studies reported significant favorable effects of n-3 PUFA on BMD or bone turnover markers. Of these, three delivered n-3 FA in combination with high calcium foods or supplements. Five studies reported no differences in outcomes between n-3 PUFA intervention and control groups; one study included insufficient data for effect size estimation. Strong conclusions regarding n-3 PUFAs and bone disease are limited due to the small number and modest sample sizes of RCTs, however, it appears that any potential benefit of n-3 PUFA on skeletal health may be enhanced by concurrent administration of calcium.
A systematic review of omega-3 fatty acids and osteoporosis. 2012
Polyunsaturated fatty acids and their relation with bone and muscle health in adults. 2013
