Osteoporosis, Inflammation, and Aging

Ginaldi Lia, Mengoli Lucia Paola, Sirufo Maria Maddalena, andDe Martinis Massimo

AbstractOsteoporosis is substantially an age-related condition characterized by low bonemass and increased bone fragility, putting the patients at risk of fractures, whichare major causes of morbidity and mortality in older people. Although aging andestrogen deficiency are probably the two most important risk factors, osteoporosiscan occur in any age of life. There are a large number of risk factors for thedevelopment of senile osteoporosis. Osteoporosis is currently attributed to vari-ous endocrine, metabolic, and mechanical factors. However, recent discoveriessuggest that these risk factors could exert their effects through immunologicallymediated modulation of bone remodeling. Emerging clinical and molecularevidences suggest that inflammation exerts significant influence on bone turn-over, inducing osteoporosis. Currently, growing understanding of bone physiol-ogy suggests that factors involved in inflammation are linked with those criticalfor bone remodeling process. Numerous proinflammatory cytokines have beenimplicated in the regulation of osteoblasts and osteoclasts, and a shift towards anactivated immune profile has been hypothesized as important risk factor. Chronicinflammation and the immune system remodeling characteristic of aging maybe determinant pathogenetic factors. Inflamm-aging itself plays a role in boneremodeling through proinflammatory cytokines, together with other morerecently discovered immunological mediators and transcription factors. Senileosteoporosis is an example of the central role of immune-mediated inflammationin determining bone resorption.

G. Lia (*) • M.L. Paola • S.M. Maddalena • D.M. MassimoDepartment of Life, Health, & Environmental Sciences, University of L’Aquila, L’Aquila, Italy

Department of Internal Medicine and Public Health, University of L’Aquila, L’Aquila, Italye-mail: lia.ginaldi@univaq.it; luciapaola.mengoli@aslteramo.it; maddalena.sirufo@gmail.com;demartinis@cc.univaq.it

# Springer International Publishing AG 2018T. Fulop et al. (eds.), Handbook of Immunosenescence,https://doi.org/10.1007/978-3-319-64597-1_64-1

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KeywordsOsteoporosis • Inflammation • Senescence

ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Inflammation and Osteoporosis: Clinical Links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Immune Regulation of Bone Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5The Immune-Skeletal Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Immunosenescence and Osteoporosis Share Similar Immune Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Regulatory Immune Mechanisms: The Cytokine Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Osteoporosis and Immune-Mediated Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Immune-Mediated Postmenopausal Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Osteoporosis and Inflamm-Aging: A Gender Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20The Immune Genetic Background of Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Introduction

Osteoporosis is a systemic pathology of the skeleton characterized by loss of bonemass, decreased bone mineral density, and loss of microarchitectural integrity, lead-ing to increased fragility and consequent risk of fractures. It is commonly consideredan age-related disorder, representing amajor cause ofmorbidity andmortality in olderpeople, together with other age-related diseases, such as atherosclerosis and neuro-degenerative disorders. Actually, in most developed countries, the human life span isgreatly increased and osteoporosis is therefore becoming an emerging public healthproblem. Osteoporosis is fundamentally an asymptomatic condition until the appear-ance of a bone fracture presenting itself as a complication with clinical visibility andoften life-threatening, similar to the tip of an iceberg whose economic costs regardingpublic health care and rehabilitation are often incisive. Everything before the fracturehas remained long unknown and it is only recently that the better understanding ofbone physiology is clarifying its pathogenesis.

Osteoporosis is viewed as a heterogeneous condition which can occur in any ageof life and its etiology is attributed to various endocrine, metabolic, and mechanicalfactors (abnormalities of parathyroid hormone and calcitonin secretion, insufficientvitamin D and calcium intake, postmenopausal hormonal condition, pregnancy,nutritional disorders, immobility, and consumption of drugs such as cortisone,among others) (Janeway and Medzhitov 2002). Aging and estrogen deficiency areprobably the two most important risk factors in developing senile osteoporosis.Currently, the emerging discipline of osteoimmunology is providing a new readingregister of senile osteoporosis in the light of immunosenescence and inflamm-aging.In this chapter we will focus on the interaction between bone and immune system,considering osteoporosis as an immune-mediated disease with a chronic inflamma-tory background.

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Inflammation and Osteoporosis: Clinical Links

Recently, growing understanding of bone physiology suggests that factors involvedin inflammation are closely linked with those critical for bone remodeling process,supporting the theory that immunosenescence significantly contributes to theetiopathogenesis of osteoporosis. But can we really consider senile osteoporosis asan immune-mediated disease or at least the result of an inflammatory process? Dailyclinical practice provides the first answers and the new concept of an immune-mediated mechanism at the basis of osteoporosis is clearly emerging. In particular,chronic inflammation and the immune system remodeling characteristic of variousimmunological diseases commonly associated with osteoporosis may be determi-nant pathogenetic factors.

For example, we can often verify coincidence of systemic osteoporosis withperiods of systemic inflammation as well as colocalization of regional osteoporosiswith areas of regional inflammation (De Martinis et al. 2006; Ginaldi et al. 2005b;Goldring 2003; Yudkin et al. 1999). In the postmenopausal period there is coinci-dence of inflammation with osteoporosis (Weitzmann and Ofotokun 2016). There isan increase in the risk of developing osteoporosis in various inflammatory conditions(Bultink et al. 2005; Haugeberg et al. 2004; Mikuls et al. 2005). Immunologicaldysfunctions, autoimmune and chronic inflammatory diseases (Loucks and Pope2005; Vikulina et al. 2010; Kotake et al. 2001), HIV infection (Annapoorna et al.2004; Mondy and Tebas 2003), hyper IgE syndrome (Leung and Geha 1988),inflammatory bowel diseases (Klontzas et al. 2016), rheumatic disorders, such asrheumatoid arthritis (Jensen et al. 2004), and lymphoid neoplastic diseases(Abrahamsen et al. 2005), in particular myeloma for the B lineage and adult T-cellleukemia lymphoma for the T lineage, are associated with osteoporosis.

Erosions seen in conditions such as rheumatoid arthritis, ankylosing spondylitis,and psoriatic arthritis are typically associated with inflammation in the joints.Proosteoclastic cytokines, such as tumor necrosis factor (TNF)-α, interleukin-1(IL-1), and IL-6, are elevated in these conditions (Ishihara and Hirano 2002) andlocal cytokine profile is consistent with the cytokines that modulate bone resorption(Bennermo et al. 2004; Mosken et al. 2005). An association between circulatinghigh-sensitive C reactive protein (hsCRP) level and bone mineral density (BMD) hasbeen observed in several immune and inflammatory diseases, as well as in healthyindividuals, suggesting a relationship between subclinical systemic inflammationand osteoporosis (Gianesan et al. 2005; Koh et al. 2005). The mechanisms linkinghsCRP and bone metabolism are not clear, but activated inflammatory cytokines arelikely involved. Inflammatory processes can upregulate many cytokines, such asIL-1, IL-6, and TNF-α, which strongly stimulate CRP production from the liver(Wang et al. 2002; Ylmaz et al. 2003) as well as induce bone resorption and decreaseBMD, measured at femoral neck and lumbar spine using dual energy X-ray absorp-tiometry (Morgolis and Wimalawansa 2006) or ultrasonographic densitometry atcalcaneus or wrist (Adami et al. 2003). In support of this hypothesis, the productionof IL-1, IL-6, and/or TNF-α by peripheral blood monocytes is positively correlatedwith bone resorption or spinal bone loss in healthy pre- and postmenopausal (Cohen-

Osteoporosis, Inflammation, and Aging 3

Solal et al. 1993; Salamone et al. 1998) women, and serum IL-6 concentrationpredicts femoral bone loss in healthy postmenopausal women (Scheidt-Nave et al.2001). In addition, serum concentrations of IL-6 and TNF-α are positively correlatedwith serum hsCRP levels in healthy subjects (Yu and Wang 2016) and those withmyocardial infarction (Ridker et al. 2000). Similarly, there is a significant inversecorrelation between erythrocyte sedimentation rate values and T-score, even in theabsence of overt diseases. The T-score is an evaluation index of bone mineral densityrepresenting the difference in standard deviations from the mean value for normalyoung adults (Morgolis and Wimalawansa 2006).

Rheumatoid arthritis (RA) is a typical example of the link between inflammationand osteoporosis. Bone loss in RA occurs both in the joints and throughout theskeleton as a result of the release of proteinases (metalloproteinases) and pro-inflammatory cytokines (IL-1, TNF-α), which are responsible for cartilage andbone destruction. As a result, disease activity is an independent risk factor forosteoporosis in RA (Jensen et al. 2004; Saidenberg-Kermanac et al. 2002).

Particularly interesting, although less immediately evident, is the link betweenimmunity and osteoporosis in advanced age, in which other well-known causesof bone resorption are also present, for example dysmetabolisms, decreased level ofsexual hormones, nutritional deficits, decreased physical activity, age-related dis-eases, hyperparathyroidism, consumption of bone resorbing drugs, etc. (Teng et al.2000). However, a more careful reading of osteoporosis reveals how the peculiarage-related immune system remodeling itself represents the most important patho-genetic factor for senile osteoporosis too.

Inflamm-aging, i.e., the chronic inflammatory status which characterizes aging(Franceschi et al. 2000; Ginaldi et al. 2005a), represents the background underlyinga wide range of age-related diseases which share an inflammatory pathogenesis.Numerous studies have shown that many cytokines, including IL-6, TNF-α, andIL-1, are elevated during senescence, and play direct roles in the pathogenesis ofosteoporosis too (Brunsgard and Nanes 2002; Nanes 2003; Brunsgard and Pederson2003).

Recently, novel insights into the crosstalk between immune and bone cells havebeen gained and a new landscape of bone remodeling is emerging, in which boneformation and resorption coexist in a dynamic equilibrium that is strictly controlledby the immunological signals (Greenblatt and Shim 2013). On the other hand, boneand immune system are functionally integrated through complex homeostatic net-works. Osteoporosis could be therefore considered a chronic immune-mediatedinflammatory disease which shares a similar immunological background withother inflammatory conditions and age-related disorders (Takayanagi 2012). Dis-coveries in this field definitely highlighted the interdisciplinarity of osteoporosis,suggesting the potential for developing new therapeutic strategies for the treatmentof osteoporosis.

Bone cells take part in various immunological functions, being variously involvednot only as target but also as players in several pathological conditions affecting notonly the immune function but also other systems and organs which are functionallyinterconnected. Therefore, the interdisciplinary field of osteoimmunology is now

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expanding beyond bone and immune cells. Senile osteoporosis is a paradigmaticexample of this kind of complex homeostatic networks.

Immune Regulation of Bone Turnover

Osteoblasts (OBs), specialized in new bone formation, are the precursors of thestructural cells of the bone, that is the osteocytes (OCys). Osteoblasts in turn derivefrom a mesenchymal stem cell (MSC) that can also differentiate into bone-marrowstromal cells and adipocytes. Osteoclasts (OCs) are on the contrary multinucleargiant cells specialized in bone resorption by the production of lysosomal enzymes(Teitellaum 2000). They stem from a myeloid precursor which also gives rise tomacrophages and dendritic cells, which are antigen presenting cells (Siggelkow et al.2003; Teng et al. 2000).

RANK receptor on OC, through the adapter protein tumor-necrosis-factor-recep-tor-associated factor 6 (TRAF6), activates NF-kB and other transcription factors,such as MAPKs, c-fos, activator protein 1 (AP1), up to nuclear factor of activatedT-cells (NFATc1), the hub of various immunological signaling pathways. At thesame time, the phosphorylation of adaptor proteins associated with distinct Ig-likereceptors, such as the immunoreceptor tyrosine-based activation motif (ITAM) andFc-receptor common gamma (FcRγ) subunit, occurs and the nuclear NFATc1becomes activate, inducing OC differentiation and proliferation (Li et al. 2014).

OCys are long-lived cells within the bone matrix performing several functions,including the control of bone remodeling. They utilize autophagy to remove dam-aged organelles and macromolecules, and thereby maintain function. When thedamage is excessive, cell death (apoptosis) pathways counteract the impact ofpotential osteocyte dysfunction on the skeleton (Jilka and O’Brien 2016). OCyapoptosis is critical in the recruitment of OCs to specific sites in response to skeletaldamage, estrogen deficiency, and other conditions that require bone removal. Signalsemanating from dying OCys stimulate neighboring viable OCys to produce osteo-clastogenic cytokines.

The main signaling pathway in bone resorption is mediated by the stimulation ofreceptor activator of nuclear factor-kB (RANK) on OCs and their precursors by itsspecific ligand RANKL, predominantly expressed on osteoblasts and stromal cells.This receptor system pertains to TNF-family molecules and is essential for thedevelopment and activation of osteoclasts. A central role in this system is alsoplayed by the ligand osteoprotegerin (OPG), competitive inhibitor of RANKL,also known as osteoclastogenesis inhibitory factor, which functions as a solubledecoy receptor to RANKL (Saidenberg-Kermanac’h et al. 2004a, b; Saidenberg-Kermanac et al. 2002). Inhibition of RANKL function via OPG prevents bone loss.Other costimulatory immune receptors also exist, for example, osteoclast-associatedreceptor (OSCAR), triggering receptor expressed in myeloid cells (TREM-2), andothers (Atchins et al. 2003; Fazzalari et al. 2001; Grimaud et al. 2003). These factorsact cooperatively with RANKL in enhancing osteoclastogenesis. Intriguingly,immune cells also express RANKL. In the immune system, RANKL is expressed

Osteoporosis, Inflammation, and Aging 5

by activated T-cells, B-cells, and dendritic cells. Therefore activated T-lymphocytescould directly induce osteoclastogenesis through RANKL (Khosola 2001; Romaset al. 2002; Takayanagi 2012).

Current evidence supports a model whereby the apoptotic demise of osteocytesinstructs neighboring viable osteocytes to synthesize cytokines such as RANKLand VEGF, which in turn recruit OCs to remove dead cells and initiate the matrixremodeling (Jilka 1998; Jilka et al. 2013; Jilka and O’Brien 2016). OCys are nowrecognized as a major orchestrator of skeletal activity, capable of sensing andintegrating mechanical and chemical signals from their environment to regulateboth bone formation and resorption. Communication within these networks isboth direct (via cell-cell contacts at gap junctions) and indirect (via paracrinesignaling by secreted signals). Moreover, the regulatory capabilities of OCysextend beyond bone to include a role in the endocrine and immune control(Schaffler et al. 2014).

In the complex scenario of osteoimmunology, that is the immune regulation ofbone turnover, the T-lymphocyte has the main role (Taubman and Kawai 2001;Willamson et al. 2002). The skeleton is physiologically in a state of dynamicequilibrium between new bone formation mediated by osteoblasts and resorptionmediated by osteoclasts. Both these processes are finely tuned by cytokines andgrowth factors. Dendritic cells, specialized to present antigens, and osteoclasts,specialized to resorb bone, share the same bone-marrow precursors of the monocytelineage and exhibit parallel lifecycles, regulated by a variety of cytokines. Release ofcells into the circulation from the bone-marrow and homing from the blood stream toperipheral tissues where the immature osteoclast precursors (OCPs) differentiate intomature osteoclasts are complicated processes involving adhesion molecules, cyto-kines, and chemokines. OCPs migrate along chemokine gradients. Stromal cell-derived factor-1 (SDF-1) produced by bone-marrow stromal cells and endotheliumhas chemotactic effects on OCPs. Transforming growth factor-β (TGF-β) down-regulates the expression of SDF-1. In chronic inflammatory conditions increasedcytokine levels in blood may feedback to bone-marrow to stimulate the egress ofmyeloid/OCPs. A major function of OCPs is to serve as a pool of progenitors fordownstream effector cells, depending upon the cytokines and growth factors impli-cated. They differentiate into CD11c + dendritic cells in the presence of granulocyte/monocyte-colony stimulating factor (GM-CSF) plus IL-4 but form tartrate resistantacid phosphatase (TRAP) + osteoclasts if exposed to RANKL (receptor activator ofnuclear factor κB ligand) and macrophage-colony stimulating factor (M-CSF)(Khosola 2001; Romas et al. 2002). The dendritic cells produce cytokines andchemokines directly or activate T-lymphocytes to indirectly promote osteoclastsand inflammation.

Many other receptor systems cooperate with RANK. EphB2 on OC functions asinhibitor receptor for RANK signal when stimulated by EphB4 on OB. Interactionbetween EphB2-expressing OC and EphB4-expressing OB facilitates the transitionfrom bone resorption to bone formation during bone remodeling. EphB4 activationon OB contemporaneously favors the coupling of bone formation and resorption byinducing osteogenetic regulatory genes (Matsuo 2010).

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The canonical Wnt/β catenin pathway, involved mainly in the response tomechanical load, promotes differentiation, proliferation, and activity of OB andinhibits their apoptosis. It encompasses a family of proteins such as Frizzled (Fz)proteins and low-density lipoprotein related receptors (LRP-5, LRP-6) which stabi-lize the β-catenin substrate regulating the transcription of target genes to induce OBdifferentiation and bone formation. WNT signaling also inhibits MSC commitmentto the chondrogenic and adipogenic lineages enhancing differentiation along OBlineage. WNT–β-catenin signaling in OB and OCy also indirectly suppressesosteoclastogenesis by increasing OPG production. The bone morphogenic protein(BMP) pathway acts as a Wnt associative stimulator whereas Dickkopf Homolog-1(DKK-1), the secreted frizzled related protein (sFRP), and the sclerostin, synthesizedby the SOST gene, act as natural inhibitors of the Wnt system (Klontzas et al. 2016).The inflammatory cytokine TNF-a reduces osteoblastogenesis by stimulatingDKK-1 and Sost expression (Weitzmann 2014).

The Immune-Skeletal Interface

The imbalance between bone formation and bone resorption, which is driven by thephysiological aging process, is also exacerbated by several pathological age-relatedconditions. The immune system exerts protective effects on the skeleton, beingessential for the physiological bone remodeling, but might also exert deleteriouseffects in several pathophysiological states, in which excessive or dysregulatedimmune functions intervene. The immune system affects both basal and pathologicalOC-mediated bone resorption. In addition, immune response also extends to theregulation of OB bone formation (Van Offel et al. 2002; Weitzmann 2014).

Following antigen recognition, T-cells become activated and produce RANKLthat induces OC differentiation and activation. Both these processes could bedownregulated by the decoy receptor OPG. In addition, they produce inflammatorycytokines, such as TNF, IL-1, IL-6, which induce osteoblasts to further expressRANKL. All of these lead to an imbalance between bone formation and resorption,with consequent osteoporosis.

OBs not only play a central role in bone formation by synthesizing multiple bonematrix proteins, but regulate OC maturation by soluble factors and cognate interac-tion, resulting in bone resorption. OC maturation requires stimulation by RANKLexpressed on OBs, and cognate interaction mediated by firm adhesion viaintercellular adhesion molecule (ICAM)-1. Proinflammatory cytokines such asIL-1 and TNF-α favor bone resorption via the induction of RANKL and ICAM-1on OBs. These inflammatory signals originate from the immune system, and suchimmunological signals to the bone are transmitted primarily via osteoblasts to induceOC maturation, resulting in secondary osteoporosis (Tanaka et al. 2005; Tanaka et al.2015). As a consequence, there is an increased stromal/osteoblastic cell-inducedosteoclastogenesis during aging. Also stromal/osteoblastic cell expression ofM-CSF, in association with RANKL, regulates osteoclastogenesis.

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Aging is accompanied by decreased OPG and increased TNF-α, IL-1, RANKL,and M-CSF expression; increased stromal/osteoblastic cell-induced osteoclas-togenesis; and expansion of the OCP pool. These changes correlate with age-relatedalterations in the relationship between OBs and OCs in bone (Cao et al. 2005).Recently, it has become evident that the activity of immune cells affects the balanceof bone mineralization and resorption carried out by the opposing actions of OBs andOCs. For example, increased bone resorption resulting in lytic bone lesions andosteoporosis is observed in many inflammatory and autoimmune diseases. Bonedestruction is also common in many cancers, both those that reside in the bone likeleukemias and multiple myeloma, and those that metastasize to the bone such asbreast and prostate cancers (Khosola 2001; Rodan and Martin 2000; Tarlow et al.1993). Dendritic cells (DCs), specialized to present antigens, and osteoclasts, spe-cialized to resorb bone, exhibit parallel lifecycles. DCs arise from multipotent pre-cursors of the monocyte lineage and are essential organizers of immune responses.They are highly specialized cells that capture antigens in peripheral tissues, migrateto lymphoid organs, and organize T-cell responses (Bancherau and Ste Bruunsgard2002). OCs are derived from the same precursors in response to interactions withosteoblasts and other bone stromal cells. Upon differentiation into mononuclear OCsand subsequent maturation and fusion into multinucleated cells, OCs actively resorbbone (Tyagi et al. 2012). These processes are dependent on a variety of cytokines,transcription factors, and inflammatory mediators. The parallel lifecycles of thesemyeloid-derived cells has led to the observation of many molecular and cellularinteractions between the bone and the immune system, which has been termedosteoimmunology (Arron and Choi 2000).

T-cells can be divided into Th1 or Th2 cells, depending on the cytokines theyproduce, Th1 producing mainly IFN-γ and IL-2 and Th2 producing primarily IL-4/IL-5/IL-10. In addition, regulatory T-cells (Tregs, CD4 + CD25 + Foxp3+)potently inhibit the function of effector T cells. Another subset of IL-17-producingeffector T helper cells, called Th17 cells, produce IL-17, IL-17F, and IL-22. Th17cells sustain inflammatory reactions and support OC formation mostly through theexpression of IL-17, which induces both RANK expression on OC precursors andRANKL production by cells supporting OC formation (Alberti et al. 2006; Alswat2017). IL-17 recruits and the activates immune cells, leading to the release of otherproinflammatory cytokines, such as IL-1 and TNF-α, therefore increasing inflam-mation and RANKL expression and synergizing with RANKL to maximize OCformation. Moreover, Th17 cells also express RANKL and can be thereforeconsidered an osteoclastogenic Th subset as well as other activated T-cellsexpressing high RANKL levels, able to directly induce OC differentiation (Moriet al. 2015).

Several immune receptors and cytokines are variously implicated in boneremodeling, some of them exerting inhibitory action, others stimulatory functionon bone resorption. The receptor for advanced glycation end products (RAGE), amember of the immunoglobulin super-family transmembrane proteins, is implicatedin inflammatory responses and in the pathogenesis of various diseases, includingage-related diseases such as diabetic complications and neurodegenerative disorders.

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RAGE and its ligands play important role in innate immune response and are alsoimplicated in OC activation and bone remodeling (Yun and Lee 2004).

Many signaling pathways are shared between the immune system and bone.Immunoglobulin (Ig)-like receptors are shared receptors which amplify the NFATc1signal. The Toll-like receptors (TLR), stimulated by pathogen-associated molecularpatterns (PAMP), utilize TRAF6 in their cascade signaling (Kassem et al. 2015). TLRinduce the release of proinflammatory and osteoclastogenic cytokines from immunecells, leading to bone resorption stimulation. They play a role in the pathogenesis ofosteoporosis of infectious diseases.

OSCAR is an immunoglobulin-like receptor involved in the cell-cell interactionbetween OB and OC (Barrow et al. 2011) which also plays critical roles in theregulation of both innate and adaptive immune responses. DAP12 is involved in theformation and function of OC (Kameda et al. 2013). Ig-like receptors associated withFcRγ and DAP12, initially characterized in NK and myeloid cells, are also essentialpartners of RANK during osteoclastogenesis (Wu and Arron 2003).

A cascade of cytokines drives OCP homing, differentiation, and activation (Joneset al. 2011). TNF induces the expression of OSCAR and other receptors importantfor OC differentiation on the surface of monocytoid peripheral blood (Goettsch et al.2011). CD80/CD86 blocks OC formation when bound to CTLA4, a negativeregulator of T cell costimulation by monocytes, which is highly expressed on Tregsurface (Hochweller and Anderton 2005; Schett 2009).

Cathepsin K is expressed in OC, degrade type I collagen in the bone matrix, and itis also involved in the TLR9-mediated activation of DCs as well as in the expressionof inflammatory cytokines, such as IL-6, IL-23, and IL-17, which in turn promoteosteoclastic bone resorption (Yosida et al. 2002).

Semaphorin 4D, expressed on OCs, maintains the bone resorption phase of boneremodeling. Since Sema4D also regulates the activation of B cells and DCs andinhibits monocyte migration, it can be considered as an osteoimmunological medi-ator (Xing et al. 2015).

The matrix glycoprotein osteopontin (OPN) is produced by several cell typesincluding immune cells, OC, endothelial and epithelial cells. It enhances OC expres-sion of the immune receptor CD44, required for cell motility, and directly mediatesOC attachment to bone extracellular matrix, required for OCP activation. As aconsequence of bone resorption, more OPN is further released from the ECM intosurrounding bone and into the circulation, thus perpetuating local and systemicosteoclastogenesis (Musso et al. 2013).

Immunosenescence and Osteoporosis Share Similar ImmuneProfile

It is the activated immune profile which, through inflammation and inflammatorycytokine production, modulates osteoblast and osteoclast activity leading to osteo-porosis. In many pathological and paraphysiological conditions, maintenance andamplification of inflammatory reactions lead to osteoclastogenesis and increased risk

Osteoporosis, Inflammation, and Aging 9

of fractures. The inducer cells in this process are immune cells, such as activatedmacrophages and lymphocytes, which produce cytokines and soluble mediators ableto stimulate osteoclast differentiation and activation. Molecules that regulateosteoclastogenesis are in fact key factors in many immunological functions.

Immunosenescence is the consequence of the continuous attrition caused by life-long antigenic load which is responsible for the chronic immune system activationand hyperproduction of proinflammatory cytokines. Therefore osteoporosis andimmunosenescence share the same immunological cell and cytokine mediators.

Thymic T-cell production declines rapidly with advancing age, conditioning theperipheral immune phenotype of elderly people and subjects with senile osteoporo-sis. Moreover, multiple mechanisms, including antigen-driven clonal expansion andhomeostasis-driven autoproliferation of postthymic T-cells, impose replicative stresson T-cells and induce the biological program of cellular senescence with character-istic phenotypic changes.

T-cell immunosenescence is associated with profound changes in T-cell func-tional profile and leads to accumulation of CD4 + T-cells which have lost CD28 buthave gained killer immunoglobulin-like receptors (KIRs), markers of natural killercells. They also exhibit cytolytic capability and produce large amounts of pro-inflammatory cytokines (Weitzmann and Pacifici 2005; Weyand and Goronzy 2004).

The increased production of proinflammatory cytokines with aging derives from achronic hyperactivation of macrophages and dendritic cells, as well as memory andsenescent T-cells. These cytokines induce expansion of OCPs which in turn maycontribute to the maintenance of inflammation through their capability to produceproinflammatory cytokines themselves and recruit other inflammatory cells, render-ing the inflammation chronic.

Osteoclastogenesis and inflammation are directly proportional to OCP levels inthe peripheral blood (Saidenberg-Kermanac et al. 2002). Characteristic of an agedimmune profile is the accumulation of activated memory cells expressing RANKL,preferentially resident in the bone and secreting osteoclastogenic proinflammatorycytokines. Therefore, through inflammation and its mediators the immune systeminfluences not only the immunological defense reactions, but each organ in the body,including bone.

The immunophenotypical analysis of peripheral blood lymphocyte subsets con-firms the deep involvement of the immune system in bone remodeling. CD3+T-lymphocytes are increased in osteoporotic patients, as well as their CD4+/CD8+ratio (Ima et al. 1990; Rosen et al. 1990), whereas CD20+ B lymphocytes aresignificantly decreased.

An expansion of the CD8 + CD56+ lymphoid subset has also been described(Kaech and Ahmed 2001). These are killer/effector lymphocytes producing largeamounts of the inflammatory cytokine TNF-α.

Finally, in osteoporotic patients there is an increase in CD45RO+ memorylymphocytes, whereas the CD45RA+ naive subset is markedly decreased (Effros2004). Based upon their homing characteristics, cytokine production, and effectorfunctions, memory T-cells have been further subdivided into central memory and

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effector memory T-cells (Gianesan et al. 2005; Kaech and Ahmed 2001; MoserBoloetscher 2001; Schluns and Lefrancois 2003). These subsets are identified by thepresence and absence of a set of cell surface markers. CD8+ effector memory T-cellsare further subdivided into two subsets, T-effector memory CD45RA negative andT-effector memory CD45RA positive, whereas CD4+ effector memory cells areprimarily CD45RA negative and only few cells are CD45RA positive.

During aging the number of central memory CD8+ T-cells is significantlyreduced, whereas the number of effector memory CD45RA positive CD8+ T-cellsis increased (Gupta et al. 2004, 2005, 2006; Saurwein-Teissi et al. 2002). Thesememory cells are mainly senescent and proinflammatory cells, able to secrete largeamounts of proinflammatory cytokines involved in the regulation of bone turnover(Sallusto et al. 2004). These findings are particularly interesting if we consider thatthe same immune profile (accumulation of activated cells and memory/effectorlymphocytes secreting proinflammatory cytokines) characterizes not onlyimmunosenescence, but also other peculiar immunological conditions notably asso-ciated with osteoporosis, such as chronic viral infections, AIDS, rheumatoidarthritis, etc.

Another important mechanism which could link inflamm-aging and osteoporosisis the regulation of immune functions by T-regulatory cells (Tregs). The role ofintrathymically generated CD4 + CD25+ regulatory T-cells in the control of allergyand asthma is well known (Akbari et al. 2003). Antiinflammatory, antiproliferative,and antiautoreactivity Tregs express innate immunity receptors and respond toproinflammatory signals and products of inflammation (Zon et al. 2005). Suchnatural regulation of Treg by immune responses to nonself may well explain thealarming epidemiology of allergic and autoimmune diseases in wealthy societies,where a variety of childhood infections have become rare or absent (Buckner andZiegler 2004; Coutinho et al. 2005). Suppression through natural or professionalCD4 + CD25+ Tregs is primarily cell-contact-dependent but is subsequentlyfollowed by cell-contact-independent T-cell inhibition mediated by second-generation T-regulatory cells (Tr1 and Th3) via the soluble factors IL-10 andTGF-β (Moser Boloetscher 2001; Stassen et al. 2004). Both these cytokines areable to antagonize immune-mediated bone resorption.

Thymic dysfunction which accompanies aging could compromise Treg genera-tion and maturation, facilitating inflammatory processes and osteoporosis (Maggiet al. 2005). Some authors described an increase in CD4 + CD25high regulatoryT-cells during aging (Gupta et al. 2005; Toraldo et al. 2003; Gregg et al. 2005),which however are quite dysfunctional. Suppressive activity of Treg cells declineswith age (Trzontwski et al. 2006) probably because of age-dependent thymic atrophyor the senescent peripheral environment. Mature and activated dendritic cells,characteristic of the senescent immune profile, produce proinflammatory cytokines,including IL-6, which render responder T-cells refractory to the suppressive effect ofTregs (Kabelitz et al. 2006).

Advancing age and loss of bone mass and strength are closely linked. Withadvancing age, the imbalance between the amount of bone resorbed by OCs and

Osteoporosis, Inflammation, and Aging 11

the bone deposited by OBs leads to bone loss and osteoporosis. Thus, aging andosteoporosis are intimately linked.

The decline in whole bone strength is due to reductions in trabecular and corticalbone density, decreased cortical thickness, and a marked increase in cortical porosity.Loss of cancellous bone mass starts in the third decade of human life while corticalbone begins to decline after the age of 50. In women, the loss of bone occurs at afaster rate after the menopause, due to the adverse role of estrogen deficiency onbone, contributing to the acceleration of skeletal involution with age. Several factorscontribute to the defective OB number in the aged skeleton: decreased number ofmesenchymal stem cells, increased apoptosis, and defective proliferation/differenti-ation of progenitor cells or shift of these progenitors toward the adipocyte lineage(Almeida 2012).

Signaling pathways that impact longevity and several diseases of aging mightalso contribute to age-related osteoporosis. Elevated OB and OCy apoptosis anddecreased OB number and activity characterize the age-related skeletal changes.Similar to other tissues, oxidative stress increases in bone with age and reactiveoxygen species (ROS) heavily impact bone. FoxOs, sirtuins, and the p53/p66 shcsignaling cascade affects bone remodeling via both ROS-dependent and ROS-independent mechanisms. p53/p66 shc signaling activation increases OB and OCyapoptosis in the aged skeleton and decreases bone mass. FoxO activation in OBsprevents oxidative stress to preserve skeletal homeostasis and attenuate Wnt/T-cellfactor transcriptional activity and OB generation. ROS/FoxO suppression of Wntsignaling may be also the mechanism by which lipid oxidation contributes to thedecline in OB number and bone formation that occurs with aging. Lipid oxidation, inaddition to its role in the development of atherogenesis, is therefore also implicatedin the pathogenesis of osteoporosis (Almeida 2012).

An aberrant NF-kB signaling contributes to the development of senile osteo-porosis. The NF-κB pathway is implicated in aging, in association with IGF-1,mTOR, SIRT1, and p53. NF-κB transcriptional activity is upregulated in a varietyof tissues with aging in response to accumulated DNA damage which drives tissuedegeneration. Another factor contributing to increased NF-κB activity with agingis the proinflammatory senescent phenotype consisting of increased expression ofIL-6, IL-8, IL-7, MCP-2, MIP-3, ICAM, Il-1α, and Il-β (Almeida 2012; Theill et al.2002).

The leading immune mechanisms involved in the pathogenesis of senile osteo-porosis are summarized in Table 1.

Table 1 Immune mechanisms involved in the pathogenesis of senile osteoporosis

Memory/effector and senescent cell expansion ROS-induced NF-kB hyperactivation

Activated macrophage and dendritic cells Osteoblast and osteocyte apoptosis

Proinflammatory and osteoclastogenic cytokinesecretion

Osteoclast precursor expansion

Impaired Treg function Osteoclast differentiation andactivation

12 G. Lia et al.

Regulatory Immune Mechanisms: The Cytokine Network

Changes in the cytokine milieu are major characteristics of aging process as well asof age-related diseases (Bennet et al. 2005). The remodeling of the cytokine networkis the hallmark of inflamm-aging (Franceschi et al. 2007). There is a complexnetwork linking the different cytokines involved in immune-mediated boneremodeling. Lymphocyte activation does not always lead to osteoporosis, the finalresult depending on the specific cytokines produced and their reciprocal interactions(Zhou and Xiong 2011). There are stimulators and inhibitors of bone resorption.These factors may elicit their effects directly, by acting on the osteoclast precursoror mature cells, such as RANKL, TNF-α, IL-1, and prostaglandin E2 (PGE2), orindirectly, via another cell type, in most cases to modulate RANKL/OPG expression,for example, parathyroid hormone-related peptide (PTHrP), PGE2, IL-11, IL-17(Arai et al. 2004; Bennet et al. 2005; Calvi et al. 2003; Kato et al. 2002; KnotheTate et al. 2004).

The main cytokines involved in bone remodeling are listed in Table 2.For example, TNF-α has the potential to regulate OC differentiation and function

in a number of ways. It may promote osteoclastogenesis indirectly through theinduction of the expression of RANKL and colony-stimulating factor- 1 (CSF-1)in bone-marrow stromal cells and bone-lining cells (Arai et al. 2004). AlternativelyTNF-α may act directly on the OCPs to promote OC differentiation. TNF-α mayfunction to increase the CD11b + OCP cell population (Joscen et al. 2000).

The proinflammatory cytokine IL-1 signals through its receptor IL-1R1. Thisinteraction is inhibited by the presence of the soluble antagonist IL-1Ra, whichcompetes with IL-1 for binding to the IL-1R1. In the presence of CSF-1, IL-1 can actdirectly to promote the fusion of mononuclear OCPs to form OCs (Kim et al. 2005)and can promote the survival and function of mature OCs. Like TNF-α, the capacityof IL-1 to promote immune response in inflammatory arthritis has made IL-1 a targetfor therapeutic blockade. The approved therapeutic agent for blockade IL-1 signal-ing in RA is a recombinant form of IL-1Ra (Cremer et al. 2002; Gupta et al. 2005;Hofbauer et al. 1998; Kim et al. 2005; Weyand et al. 2003; Wiethe et al. 2003), andits use has proved efficacious in the treatment of inflammatory arthritis with retar-dation of focal bone erosion in a significant number of patients.

IL-4 is one of the inhibitor cytokines. Moreover, its hyperproduction character-izes an atopic background and stimulates IgE synthesis. Interestingly, in some cases,

Table 2 Main cytokines involved in bone remodeling

Osteoclastogenesis stimulators Osteoclastogenesis inhibitors

TNF-α IL-15 IL-4 IL-18

IL-1 IL-17 IL-10 IL-23

IL-6 IL-31 IL-12 IL-33

IL-7 IL-32 IL-13 GM-CSF

IL-11 M-CSF TGF-β IFN-γ

Osteoporosis, Inflammation, and Aging 13

allergy and Th2 immune profile could result protective towards osteoporosis andother inflammatory diseases (Elenkov et al. 2005). A Th2-mediated atopic diseaseprotection in Th1-mediated diseases such as RA has been described (Morgolis andWimalawansa 2006). In an unpublished study, bone mineral density in allergicpatients who have not undergone cortisone therapy resulted higher compared tosex-and age-matched healthy controls. An inverse correlation between bone mineraldensity and total IgE, that are markers of atopy, also exists.

IL-6 is a key regulator of osteoclast differentiation in response to estrogendeficiency in postmenopausal bone loss. IL-6 is significantly increased duringaging and its level strongly correlates with the risk of osteoporotic fractures(Morgolis and Wimalawansa 2006). The pleiotropic proinflammatory cytokineIL-6 has been detected at elevated levels in synovial fluid and sera of RA patientswith active disease. In addition IL-6 and its soluble receptor (sIL-6R) levels in RApatients have been correlated with the degree of radiographic damage.

IL-7 is a cytokine that stimulates thymic T-cell production and induces theexpansion, activation, and differentiation of mature circulating T-cells. IL-7 inducesbone loss in vivo, presumably by stimulating the differentiation of OCPs into OCs.IL-7 also upregulates RANKL production in T-cells. IL-7 induces proinflammatoryand osteoclastogenic cytokine production and the expansion of B220+ IgM-B cellprecursors. These cells could lead to bone destruction by overexpressing RANKL or,alternatively, by differentiating into OCPs in response to M-CSF and/or RANKL.

IL-11 regulates the growth and development of hematopoietic stem cells. LikeIL-6, IL11 has been implicated in mediating osteoclast differentiation through theupregulation of RANKL expression in cells of the OB lineage.

IL-17 is a proinflammatoy cytokine secreted predominantly by activatedCD4 + CD45RO+ memory T-cells (Walsh et al. 2006). Through its ubiquitouslyexpressed receptor, IL-17R leads to the activation of the adapter molecule TNFreceptor associated factor 6 (TRAF6) and subsequent modulation of target geneexpression via signaling through the NF-kB and mitogen-activated protein tyrosinekinase pathways (Tarlow et al. 1993; Lopez and Buchman 2000). IL-17 induces theproduction and secretion of IL-1, IL-6, IL- 8, TNF-α, GM-CSF, and PGE2. IL-17also induces the expression of RANKL and decreases OPG expression in both RAsynoviocytes and cells of the osteoblast lineage (Taubman and Kawai 2001).

IL-18, a member of the IL-1 superfamily of cytokines, is present at elevated levelsin the synovial membrane, synovial fluid, and serum of RA patients. Originally,IL-18 was demonstrated in vivo to inhibit OC differentiation indirectly via theinduction of GM-CSF expression by both cells of the OB lineage and activatedT-cells. IL-18 may promote OC differentiation by inducing T-cell expression ofRANKL.

Osteopontin, also known as Eta-1 (early T-lymphocyte activation gene-1), is asecreted phosphorylated glycoprotein that functions both in inflammation and boneremodeling. Important in mediating T-helper 1 cell immune responses, osteopontinis produced by activated T-cells and macrophages. It interacts with CD44 andintegrin receptors to promote chemotaxis and migration of monocyte-macrophagecells and enhances B-cell proliferation and antibody secretion. It is also produced by

14 G. Lia et al.

both OCs and cells of the OB lineage and acts to promote cell-matrix adhesion viaintegrin (Takayanagi 2012). Interplay between interferon and other cytokine systemsis also central in bone metabolism.

TRAF6 is a crucial signaling molecule regulating a diverse array of physiologicalprocesses, including adaptive immunity, innate immunity, bone metabolism, and thedevelopment of several tissues including lymph nodes, mammary glands, skin, andthe central nervous system. It is a member of a group of six closely related TRAFproteins, which serve as adapter molecules, coupling the TNF receptor (TNFR)superfamily to intracellular signaling events. Among the TRAF proteins, TRAF6is unique in that, in addition to mediating TNFR family signaling, it is also essentialfor signaling downstream of an unrelated family of receptors, the IL-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF6 therefore represents an importanttarget in the regulation of many disease processes, including immunity, inflamma-tion, and osteoporosis (Willamson et al. 2002). There exists an intimate interplaybetween the bone and the immune system. Skeletal bone is more than a frame onwhich to hang flesh and organs, it is also the source of bone-marrow-derivedhematopoietic cells. Many myeloid lineage hematopoietic cells express receptorssuch as CD40, RANK, and TLRs, which use TRAF6 for signaling and are involvedin the generation of adaptive and innate immunity. Interferon (INF)-γ interferes withthe OC differentiation induced by RANKL, and this mechanism is critical for thesuppression of pathological bone resorption associated with inflammation.

Also antigen presenting cells (APC), in addition to stimulating bone resorption,could negatively regulate osteoclastogenesis through upregulation of the RANKLdecoy receptor OPG. The secretion of IFN-γ, in particular, appears to be crucial andmultifaceted in immune-mediated osteoclastogenesis by shifting myeloid stem celldifferentiation from OCPs to DCs. During senescence there is an impaired OPGproduction as well as an impaired IFN-γ production (Alberti et al. 2006; Ho et al.2005), contributing to a derangement of the global counter-regulatory system.

Activated T-cells exert both positive and negative control on osteoclastogenesis(Weitzmann and Pacifici 2006). In fact, in addition to the osteoclast activatorRANKL, they express IFN-γ too, which binds to its receptor on OCs. This inducesthe proteasomal degradation of transcription factor TRAF6 leading to an inhibitionof the signal transduced by RANKL and an inhibition of OC function (Teng et al.2000). This direct inhibitory effect of IFN-γ on OCs contrasts with its indirectstimulatory activity through lymphocyte activation and cytokine production. Infact, IFN-γ is also a potent inducer of expression of Class II histocompatibilitycomplex antigens on antigen presenting cells. This increases T-cell stimulationmediated by antigen receptor, inducing further immune activation, proinflammatorycytokine production, and consequent OC stimulation.

RANKL induces the INF-β gene in osteoclast precursor cells, and this inductionconstitutes a critical aspect of the negative feedback regulation mechanisms ofRANKL signaling to suppress excessive osteoclastogenesis. An important functionof signal transducer and activator of transcription I (Stat 1), the essential transcrip-tion factor for both type I and type II IFN responses, is therefore the regulation of OBdifferentiation.

Osteoporosis, Inflammation, and Aging 15

The binding of RANKL to its receptor RANK results in the recruitment ofTRAF6, which activates NF-kB and c-jun N-terminal Kinase (JNK) pathways andinduces c-Fos expression. The effect of T-cells on osteoclastogenesis thereforedepends on the balance between RANKL and IFN-γ. IFN-γ signals the cell throughactivation of the transcription factor signal transducer and activator of transcription1 (Stat 1). The active form of Stat 1, termed IFN-γ activated factor (GAF), inducestarget genes of IFN-γ either directly or through the induction of the transcriptionfactor IFN regulatory factor-1 (IRF-1) (Ho et al. 2005). During acute immunereaction, an enhanced production of IFN– counterbalances the augmentation ofRANKL expression and reduces aberrant OC formation.

Osteoporosis and Immune-Mediated Diseases

Rheumatoid arthritis, seronegative spondyloarthropathies including psoriatic arthri-tis (Yang et al. 2016), and systemic lupus erythematosus (SLE) are all examples ofrheumatic diseases in which inflammation is associated with skeletal pathology(Bultink et al. 2005; Lange et al. 2000; Vikulina et al. 2010; Walsh and Gavallese2004; Walsh et al. 2005). Although some of the mechanisms of skeletal remodelingare shared among these diseases, each disease has a unique impact on articular boneor on the axial or appendicular skeleton. RA is the prototype for an inflammatoryarthritis, in which inflammation is associated with progressive bone resorption.Several immunological findings are shared by RA and senescence, suggestingsimilar immunopathogenetic mechanisms for bone resorption in both these condi-tions (Goronzy and Weyand 2003). Patients with RA have age-inappropriatetelomeric shortening of hematopoietic precursor cells. Their output of novelT-cells from the thymus is impaired. The peripheral T-cell pool is occupied byfunctionally altered T-cells, which bear the characteristics of prematurely agedlymphocytes. Global T-cell defects include a sharp contraction in T-cell diversity,the accumulation of expanded clonotypes, and preponderance for senescent T-cellsin the T-cell compartment (Goronzy and Weyand 2005; Weitzmann and Pacifici2005; Weyand and Goronzy 2004). This immune phenotype is shared by otherpathologic conditions characterized by increased incidence of osteoporosis, suchas HIV infection. The overproduction of proinflammatory cytokines, such as TNF-α,further impairs the function of hematopoietic stem cells, aggravating the impact of agenetically determined risk factor. Recently, a new disease model for RA has beenproposed (Weyand and Goronzy 2004). Instead of restricting the biological role ofHLA-DRB1 molecules to the presentation of arthritogenic antigens, these HLAmolecules or genes in linkage disequilibrium to the B1 locus could regulate hemo-poietic stem cell biology. In HLA-DR4+ individuals, stem cells proliferate exces-sively, giving rise to prematurely aged T-cells. If combined with additionalrestrictions in thymic T-cell production, the T-cell pool becomes senescent, withrestriction in diversity and limited ability for clonal burst. The same phenomenonduring senescence is triggered by lifelong antigenic burden and thymic atrophy(Ginaldi et al. 2005b). Senescent T-cells express novel regulatory receptors, are

16 G. Lia et al.

proinflammatory, and are prone to autoreactivity, promoting chronic inflammatorylesions, such as rheumatoid synovitis and osteoporosis (Weitzmann and Pacifici2005; Weyand and Goronzy 2004). Three major forms of bone loss have beendescribed in RA: focal articular erosions, a hallmark of RA; periarticular boneloss, occurring adjacent to inflamed joints; and generalized osteoporosis, leadingto an increase in fracture risk. The synovium is the major target of the inflammatoryprocess in RA. Activated lymphocytes in the inflamed synovium overexpressRANKL and TNF-α which stimulate bone-marrow OCPs to proliferate and enterthe blood stream. In turn, activated macrophages in inflamed joints produce variouschemokines, small inflammatory chemotactic cytokines, which drive OCP migrationand homing in the periarticular bone. The elevated concentration of RANKL andTNF-α in the rheumatoid synovial fluid stimulates maturation and activation of OCswhich resorb bone. Circulating OCPs secrete inflammatory cytokines amplifyinginflammatory circuits at a systemic level (Weitzmann and Ofotokun 2016). Circu-lating OCP number has been proposed as a marker of osteoporotic risk and thera-peutic response.

TNF-α also induces OB apoptosis, decreasing bone formation. Histologic exam-ination of the periarticular osteoporotic region in patients with RA shows functionalFas expression and apoptosis in OBs. IL-1β and TNF-α regulate OB cell number byupregulating the Fas-mediated apoptosis of OBs (Tsaknaridis et al. 2003). Thedefective clearance of apoptotic cells is associated with autoimmunity and inflam-mation (Savil et al. 2002). Under normal conditions, clearance of apoptotic cells byphagocytic cells is associated with secretion of antiinflammatory cytokines, includ-ing IL-10 and TGF-β1, resulting in the inhibition of inflammation. However, underpathological conditions associated with excessive apoptosis and/or decreased clear-ance of apoptotic cells, apoptotic cells may directly induce caspase-1 dependentsecretion of IL-1β and IL-8 or under secondary necrosis may induce secretion ofother proinflammatory cytokines. During aging a defective clearance of apoptoticcells as a result of poor phagocytosis by aged DCs results in secondary necrosis andrelease of endogenous ligands for toll-like receptors to activate phagocytic cells todifferentiate into more mature phenotype and secrete proinflammatory cytokines(e.g., TNF-α and IL-6) (Gupta et al. 2006). In immune-mediated osteoporosis, inaddition to systemic overproduction of bone-resorbing proinflammatory cytokines,nitric oxide and prostaglandin also play a role, mainly stimulating OB apoptosis.

All these basic immunological mechanisms have important clinical implications.In RA and psoriatic arthritis the degree of inflammation and disease activity correlatewith focal erosions and systemic osteoporosis. Bisphosphonates are drugs widelyused in the therapy of osteoporosis, able to improve BMD and decrease the risk offractures in patients with RA and steroid-induced osteoporosis (Morgolis andWimalawansa 2006). They are able to regulate cell growth and apoptosis and mayinhibit the inflammatory response of macrophages. They exert antiinflammatoryactivity by the inhibition of the release of inflammatory mediators from activatedmacrophages, such as IL-1, IL-6, and TNF-α and prevent dexamethasone-inducedgrowth retardation and apoptosis both in OBs and chondrocytes (Santini et al. 2004;Udagawa 1990; Walsh and Gavallese 2004). That the RANK/RANKL/OPG

Osteoporosis, Inflammation, and Aging 17

pathway is central to the osteoporosis pathogenesis is confirmed by the elevatedantiresorptive capacity of the anti-RANKL monoclonal antibody (MoAb)denosumab, utilized in osteoporosis therapy (Brown et al. 2014).

Blockade of the RANKL/RANK signaling pathway represents an attractivetarget for therapeutic intervention in the prevention of bone loss in RA. In initialhuman trials, the effects of OPG were examined in a cohort of postmenopausalfemales (Bekker et al. 2001). A single injection of OPG resulted in a sustainedreduction in the level of urinary N-telopeptide, a stable collagen breakdownproduct, consistent with a reduction in bone resorption activity. However, theessential role of the RANKL/RANK/OPG pathway in physiological boneremodeling would suggest that modulation, rather than complete inhibition, ofthis pathway may be the desirable aim of therapeutic intervention. The develop-ment of small molecules (or peptidomimetics) that target the RANKL/RANKsignaling pathway (molecules that mimic OPG action or modulate endogenousOPG mRNA expression) may provide a greater ability to modulate inflammation-induced osteoclast differentiation without complete inhibition of this pathway(Cheng et al. 2004; Walsh and Gavallese 2004). Blockade of TNF-α activityusing biologic agents, including recombinant soluble p75TNFR, a chimericmouse-human, and fully humanized anti-TNF-α antibodies, has demonstratedefficacy in reducing the clinical signs and symptoms of RA and in retardatingradiographic progression of focal bone erosions (Saidenberg-Kermanac’h et al.2004a; Walsh and Gavallese 2004).

Since RANKL is expressed on activated T-cells, and is crucial for T-cell-dendriticcell communication, one might expect massive bone resorption under most inflam-matory conditions. Although RANKL-expressing T-cells in chronic inflammatoryconditions such as RA and inflamm-aging can stimulate OCs leading to bonedestruction, the constant activity of T-cells fighting the universe of antigens towhich we are exposed does not usually cause extensive bone loss. As previouslyexposed, a crucial counter-regulatory mechanism whereby activated T-cells caninhibit RANKL-mediated OC development and activation is through the action ofIFN-γ. In mice deficient for the IFN-γ receptor, bone destruction in an autoimmunearthritis model is greatly exacerbated. While T-cells involved in inflammatoryresponses express RANKL, they also secrete IFN-γ. IFN-γ can block RANKL-mediated osteoclastogenesis, possibly through the activation of the ubiquitin-proteasome pathway leading to TRAF6 degradation (Willamson et al. 2002).Given the essential roles of TRAF6 in immunity and a diverse array of biologicalprocesses, it is desirable to obtain TRAF6 inhibitors to facilitate the development oftherapeutics for controlling inflammation and a wide range of diseases, such asosteoporosis and other osteolytic conditions (Takayanagi 2012).

Interestingly, despite T-cell infiltration observed in arthritic joints, IFN-γ expres-sion in these T-cells is suppressed. The paucity of IFN-γ and the enhanced expres-sion of RANKL may underlie the activation of osteoclastogenesis in arthritis. T-cellswhich infiltrate rheumatoid synovium have an expression of surface markers formemory T-cells, a low production of IFN-γ or IL-2, and hyporesponsiveness toin vitro restimulation.

18 G. Lia et al.

Therefore, not always immune activation exerts resorptive effects on bone,probably explaining the different clinical manifestations of certain immune diseases.For example, only 4–6% of patients with SLE develop erosive arthritis, despite thefrequent articular involvement on presentation (50%). The hypothesis to explain thisphenomenon is that systemic interferon-α diverts the bone-marrow-derived myeloidprecursors away from the osteoclast lineage and stimulates their differentiation intodendritic cells. In SLE patients there is an increased interferon production and anti-TNF-α therapy is scarcely effective (Ginaldi et al. 2005a). Therefore, the innateimmune TNF/IFN axis in patients with autoimmune disease dictates their erosivephenotype (Shwarz et al. 2006).

Although it is well documented that IFN-γ has a bone-protective effect in antigen-specific autoimmune arthritis, recent studies suggest that IFN-γ may have a causalrole in the bone loss associated with estrogen deficiency. Pacifici et al. propose thatIFN-γ activates antigen presentation through Class II transactivator (CIITA) induc-tion, leading to the accumulation of a TNF-α-producing T-cell population (Walshand Gavallese 2004; Walsh et al. 2005).

Immune-Mediated Postmenopausal Osteoporosis

The osteoimmunological approach suggests that activated immune cells contributeto menopausal changes in bone remodeling by producing proinflammatory cytokines(Li et al. 2014; Mansoori et al. 2016; Weinhold and Ruther 1997; Weitzmann 2013,2014; Yun and Lee 2004). Postmenopausal osteoporosis is therefore a clear exampleof the mutual influences between immune system, bone, and endocrine system. Inaddition to specific target organs (breast and reproductive system), estrogens havetheir receptors also on immune cells and bone, as well as on bone marrow precursors.Menopausal estrogen decline increases T-cell activation and proliferation by increas-ing APC activity of macrophages through increased MHCII expression and byreducing T-cell apoptosis. These actions result in the expansion of the pool ofactivated T-cells which are responsible for the chronic stimulation of OC formationand consequent bone loss (Almeida 2012; Barbour et al. 2012; Cagnetta and Patella2012; Faienza et al. 2013; Tsuboi et al. 1999; Zhao 2012).

There is progressive loss of bone tissue after natural or surgical menopause,leading to increased fractures within 15–20 years from the cessation of ovarianfunction (Riggs et al. 1998). Postmenopausal osteoporosis should be regarded asa product of an inflammatory disease triggered by estrogen deficiency, rather than amere metabolic and endocrinologic condition. OBs, OCys, and OCs express func-tional estrogen receptors. These receptors are also expressed in bone-marrow stromalcells, the precursors of OBs, which provide physical support for nascent OCs,T-cells, and B-cells. Estrogen signals through two receptors, ERα and Erβ. Bonecells contain both receptors.

Although estrogen is established to have direct effects on bone cells, recentstudies have identified additional unexpected regulatory effects of estrogen centeredat the level of the adaptive immune response (Weitzmann and Ofotokun 2016).

Osteoporosis, Inflammation, and Aging 19

Estrogens have important roles in the regulation of immune function. Ovariectomyincreases the number of TNF-producing T-cells. Estrogen deficiency results in amarked increase in proinflammatory cytokines, including IL-1, IL-6, TNF-α,M-CSF, IFN-γ, and others. Estrogen deficiency is also associated with decreasedproduction of OPG and TGF-β, which counteract bone resorption. TGF-β is apowerful repressor of T-cell activation. Estrogen deficiency upregulates IFN-γproduction through TGF-β downregulation. Generally, following an innate immuneactivation, IFN-γ functions as an antiresorptive agent. Conversely, when T-cellactivation occurs through an adaptive immune response, as in estrogen deficiency,IFN-γ stimulates bone resorption. Estrogens also repress the production of IL-7, apotent stimulator of T and B proliferation and inducer of bone destruction(Weitzmann 2014).

Reactive oxygen species (ROS) may play a role in postmenopausal bone loss bygenerating a more oxidized bone microenvironment. The NO donor nitroglycerin isalso reported to prevent bone loss in ovariectomized rats. OCs have been shown toboth generate and be activated by ROS. Glutathione peroxidase, responsible forintracellular degradation of hydrogen peroxide, is the predominant antioxidantenzyme expressed by OCs and is upregulated by estrogen. ROS are importantstimulators of antigen presentation by DC-induced T cell activation. Antioxidantspotently inhibit DC differentiation and activation of T-cells in part by suppressingexpression of MHC Class II and costimulatory molecules in response to antigen.ROS are also generated upon DC interaction with T-cells and can reduce T-cell lifespan by stimulating T-cell apoptosis. Estrogen deficiency lowers antioxidant levels,thereby increasing ROS. Additionally, estrogen deficiency augments TNF expres-sion by enhancing OC-mediated TNF production and by stimulating APC-inducedexpansion of the TNF-producing T-cells that are central to bone destruction (Konget al. 1999).

There are two transcription factors, NF-kB and AP-1, which are regulated byestrogen and control the expression of IL-12 and IL-18 in macrophages. Thestimulation of INF-γ secretion through the enhanced production of INF-γ-inducingcytokines IL-12 and IL-18 by macrophages is another mechanism by which estrogendeficiency activates immune system (Cenci et al. 2003; Gao et al. 2004; Roggiaet al. 2001).

In summary, menopause increases T-cell activation and proliferation by increasingAPC activity of macrophages through increased MHCII expression and by reducingT-cell apoptosis. These actions result in the expansion of the pool of activated T-cellsin the bone marrow which are responsible for the chronic stimulation of OC forma-tion and consequent bone loss (Hofbauer et al. 1998; Jilka 1998; Pacifici 1999;Pfeilschifter et al. 1998; Roggia et al. 2001; Tilstra et al. 2011).

Osteoporosis and Inflamm-Aging: A Gender Perspective

Human longevity, inflamm-aging, and the onset of age-related diseases, mainlyosteoporosis, are strongly influenced by gender, defined as the combination betweenbiological sexual characteristics (anatomy, reproductive functions, sex hormones)

20 G. Lia et al.

and factors related to behavior, social role, and lifestyle. Women live longer thanmen but in worse health conditions and the epidemiology of age-related diseases isdifferent between genders, often changing in women after menopause. In addition tohormonal influences, basic biological mechanisms, such as age-related X chromo-some inactivation skewing, gut microbiome changes, maternally inherited mitochon-drial DNA genetic variants, and other complex cellular mechanisms are probablyresponsible for gender differences in aging and age-related diseases (Ostan et al.2016). Gender influences the pathophysiology and clinical presentation of osteopo-rosis and the incidence of bone fractures. Osteoporosis and its complications affectboth genders but at different ages and rates. Osteoporosis is more common inwomen. Many factors, associated with both bone structure and falls, influence thedifference in fracture risk between men and women. Men develop bone loss at a laterage compared to females; however, they build larger bones with better micro-architecture and have less increase in bone resorbing during life. Women start withlower bone density and lose bone mass more quickly as they age. The menopausaldecline in estrogen levels represents a central pathogenetic factor in postmenopausaland senile osteoporosis in females. Premenopausal women have more estrogen thanmen; however, because of the dramatic drop in estrogen production during meno-pause, females are more likely to experience bone loss and osteoporosis at that time.Osteoporosis is four times more common in women than in men, but men tend tohave more osteoporosis-related complications. Gradual bone loss is common withaging in both sexes but women tend to lose bone at younger age and at a faster ratethan men. Women older than 50 years of age have a four times higher rate ofosteoporosis and tend to have fractures 5–10 years earlier compared to men.Osteoporotic fractures in males occur at higher bone density and hip fractures resultin a greater risk of death compared to females (Alswat 2017).

Gender differences also underline the pathogenetic influence of proinflammatoryprocesses sustaining osteoporosis. Inflamm-aging is manifested differently at thegenomic level in nonagenarian men and women. Transcriptomic and epigeneticanalyses reveal a gender difference in aging-associated inflammation. This genderdifference in the genomic regulation of inflammatory response seems to be, at leastin part, of epigenetic origin (Nevalainen et al. 2015). Estrogens, androgens, andprogesterone affect cells of the innate and adaptive immune system differently inmen and women and during the reproductive phase of life. There is a complexinterplay between menopause and immunosenescence and osteoporosis. Estrogendeficient bone loss is the result of complex pathways involving many differentcytokines. Inflamm-aging and menopause potentiate each other in inducing post-menopausal and senile osteoporosis (Engelmann and Messaoudi 2012). Menopausecontributes to inflamm-aging and increases the expression of osteoclastogenic cyto-kines by T cells, which in turn exacerbates postmenopausal osteoporosis.

Because osteoporosis occurs more frequently in women than men, less attentionis paid to bone health in men. For this reason, osteoporosis in men is oftenunderscreened, undiagnosed, and untreated, even when they have fractures. Menhave a shorter life expectancy compared with women and are less likely to receivemedical treatment for osteoporosis prevention and treatment even when theyundergo bone fractures (Pietschmann et al. 2009; Delmas 2002). Although more

Osteoporosis, Inflammation, and Aging 21

common in women, senile osteoporosis is increasing in men during the past fewyears. An appropriate gender-specific medicine approach to osteoporosis is thereforeurgently needed. Considering osteoporosis in a gender perspective should not meanfocusing only on women but rather, taking gender differences into account, ensuringa personalized and effective medicine.

The Immune Genetic Background of Osteoporosis

Osteoporosis could be considered an inflammatory disorder with a strong geneticcomponent. Bone mineral density is largely controlled by genetics. Proinflammatorycytokine polymorphisms are genetic markers of both inflamm-aging and osteoporosis.The genetic background which favors the onset and progression of osteoporosis isthe same that determines strong inflammatory immune responses through the hyper-production of inflammatory cytokines and/or the decreased secretion of anti-inflammatory and regulatory factors. A number of cytokine genes and genesinvolved in inflammatory responses are polymorphic and may be important fordefining the magnitude of the individual responses to a given environmental stim-ulus of cytokine production. The list includes genes that affect cytokine expression,binding of cytokines to their receptors, genes involved in the cytokine signalingpathways, and many others. There is a close link between cytokine polymorphismsand risk and severity of osteoporosis and fractures. Langdahl et al. (2000) alsoshowed that genotypes associated with a low IL-1ra production (A1A1/A3) weresignificantly more frequent in women with osteoporotic fractures compared withnormal individuals. IL-6 polymorphisms are able to influence the risk of osteoporo-sis as well as other chronic disorders involving IL-6 activity (Ferrari et al. 2003).Two promoter polymorphisms regulating IL-6 gene expression, �572 and�174 G> C, are associated with circulating levels of C-reactive protein and markersof bone resorption in postmenopausal women. For example, a single nucleotidepolymorphism in the promoter region of the IL-6 gene at position�174 (G> C) hasbeen reported to be associated with a variety of major age-related diseases whichshare an inflammatory background, as well as with osteoporosis. Individuals withthe G genotype have significantly higher plasma IL-6 values than do individualswith the C genotype. Therefore the �174 G > C single-nucleotide polymorphism inthe promoter region of the IL-6 gene is functional in vivo with an increasedinflammatory response associated with the G allele (Bennermo et al. 2004). Con-sidering the central role of IL-6 in bone resorption, this finding could have clinicalrelevance.

A relationship between the production of IL-1 and IL-6 by whole blood cells,bone mineral density, and polymorphisms in IL-1 system and IL-6 gene in postmen-opausal women has also been documented (Armour et al. 2001; Koh et al. 2005).The loci for the human IL- 1α, IL-1β, and Il-1Ra are all linked within the proximalregion of the long arm of chromosome 2. IL-1β and IL-1Ra are involved in highturnover bone loss after menopause (Chen et al. 2002). Different polymorphismshave been described in the IL-1β gene and at least two of them could influence

22 G. Lia et al.

protein production: one is located within the promoter region, the other in exon5 (Cheng et al. 2004; Chen et al. 2002; Pociot et al. 1992). Polymorphisms in theIL-1β exon 5 may influence gene transcription and protein production (Pociot et al.1992). The Taq I IL-1β exon 5 gene polymorphism is one of the candidate geneticmarkers responsible for osteoporosis in postmenopausal women, and this geneticlocus may play a central role in postmenopausal trabecular bone loss (Chen et al.2002). Five alleles of the IL-1Ra gene have been reported, corresponding to 2, 3,4, 5, and 6 copies of an 86-basepair sequence repeat located in intron 2 (Tarlow et al.1993). Bone metabolism as well as inflammatory processes are influenced by thevitamin D receptor gene (VDR). The VDR gene may be involved in BMD differ-ences, bone metabolism, and inflammatory processes in ankylosing spondylitis(Obermayer-Pietsch et al. 2003). With respect to TGF-β, a 1-base deletion in intron4 of the TGF-β1 gene has been associated with low BMD, increased bone turnover,and an increased rate of fragility fractures in osteoporotic Danish and Italian women(Ambrogini et al. 2005).

Conclusion

What is the finality of the close relationship between inflammation and boneremodeling? One possible explanation could be that bone has not only structural,but also storage function for calcium and phosphate salts and defense functions.Postmenopausal osteoporosis should be regarded as the product of an inflammatorydisease bearing many characteristics of an organ limited autoimmune disorder,triggered by estrogen deficiency and brought about by chronic mild decreases inT-cell tolerance. Why such a pathway should have emerged is intriguing. Oneexplanation is suggested by the need to stimulate bone resorption in the immediatepostpartum period in order to meet the markedly increased maternal demand forcalcium brought about by milk production. The signal for this event is the drop inestrogen levels early in the postpartum. Postmenopausal bone loss should beregarded as an unintended recapitulation of this phenomenon. Another response todelivery is the restoration of normal immune reactivity and the loss of tolerance tothe fetus. It is tempting to speculate that cessation of ovarian function induces boneloss through an adaptive immune response because natural selection has centralizedthese two key adaptations to postpartum within the immune system (Takayanagi2012; Weitzmann 2014).

Inflammatory responses require a ready supply of calcium for cellular activationand signal transmission. Also in this case, as well as in lactation during the postpar-tum, calcium derives from bone resorption. During evolution, T-lymphocyteassumed the central role of director of these complex integrated systems. In thisperspective, osteoporosis may reflect a state of disequilibrium between structuraldemand for calcium and phosphate and their biological demand during metabolicallyactive states such as inflammation (Yudkin et al. 1999). Therefore inflammationcould be considered the main force driving osteoporosis.

Osteoporosis, Inflammation, and Aging 23

An increasingly close interaction between bone and immune system, referred toas immune-skeletal interface, is recently emerging. The understanding of the inter-face between bone and immune system is essential not only for a correct clinicalapproach to osteoporosis, but also for the understanding of an increasing number ofphysiological and pathological conditions related to osteoporosis (Harsløf andLangdahl 2016). Therefore, the discovery of the existence of the immune-skeletalinterface, seen as a systemic model of integrated signaling pathways and cytokinesworking in a cooperative fashion, has brought about wide repercussions for a rangeof disease conditions beyond osteoporosis, including menopause, senescence, infec-tious, neoplastic, inflammatory, and rheumatic diseases, in which same pathogenicpathways are often shared. Thus the panorama of osteoimmunology is expanding toan ever widening interdisciplinary study, revealing important interconnections withan increased spectrum of different disease entities.

The correct understanding of the complex language existing between immunesystem and bone during aging is the essential requirement for the individualizationof new and effective therapeutic targets for both osteoporosis and inflammation.

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