Inflammatory Bowel Disease and Bone

11 Inflammatory Bowel Disease and Bone Francisco A. Sylvester1, Anthony T. Vella2 UNIVERSITY OF CONNE CTICUT SCHOOL OF ME DICINE , HARTFORD, CT, USA, UNIVERSITY OF CONNECTICUT SCHOOL OF MEDICINE, FARMI NGTON, C T, USA 1

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CHAPTER OUTLINE Introduction ........................................................................................................................................ 325 The intestinal immune system.......................................................................................................... 326 Innate immune functions and IBD pathogenesis............................................................................ 327 The adaptive immune system and IBD ............................................................................................ 328 Immunological differences between Crohn’s disease and ulcerative colitis ............................... 329 IBD and osteoimmunology................................................................................................................ 330 Effects of IBD on bone mass ............................................................................................................. 330 Mechanisms by which IBD affects bone mass ................................................................................ 331 General disease-related and treatment factors.......................................................................... 331 Disease-related immune factors................................................................................................... 332 RANKL and OPG ............................................................................................................................ 334 Conclusion ........................................................................................................................................... 335 References........................................................................................................................................... 335

Introduction Inflammatory bowel disease encompasses two main entities, Crohn’s disease and ulcerative colitis. IBD affects approximately 1.2 million Americans, and affects children and adults. Both ulcerative colitis and Crohn’s disease are associated with significant bone mass deficits. Although both cause chronic intestinal inflammation, they have important clinical and pathogenic differences that are relevant to how they affect the skeleton. Crohn’s disease is characterized by patchy inflammation of the full thickness of the bowel wall, and can affect any part of the gastrointestinal tract. Most commonly, Crohn’s disease localizes to the distal ileum and colon. Inflammation can perforate the intestinal wall and produce abscesses and fistulization into neighboring organs or the skin. Ulcerative colitis on the other hand typically produces continuous inflammation that begins in the rectum, which classically is limited to the lamina propria of the colon. Osteoimmunology. DOI: 10.1016/B978-0-12-375670-1.10011-1 Copyright Ó 2011 by Elsevier Inc. All rights reserved.

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Although both Crohn’s disease and ulcerative colitis can have extra-intestinal manifestations, Crohn’s disease tends to affect bone mass more frequently and severely than ulcerative colitis, especially in growing children [1].

The intestinal immune system To understand the relationships between the intestinal immune system and bone metabolism in IBD, we will first describe its functions in health and disease. Mouse models of IBD have been invaluable in deriving much of this knowledge. In general, these models are characterized by inflammation of the colon, which may be acute and selfresolving or chronic and non-remitting, depending on the model [2]. Many of these models depend on bacterial colonization of the intestine, in the absence of which inflammation does not develop. This underscores the importance of bowel flora in the pathogenesis of IBD, as detailed below. The intestine hosts the largest mass of immune cells in the body. Its luminal surface is exposed to the biggest load of foreign antigens. These come from multiple sources, including food, bacteria, and xenobiotics. These antigens are normally confined to the luminal surface of the intestine by a single layer of cells. This layer includes epithelial cells, goblet cells, and Paneth cells, each with specialized functions. These cells form a barrier that is both mechanical and functional. Epithelial cells adhere to each other with tight junctions. Mucus secreted by goblet cells forms a protective layer. Paneth cells secrete antibiotic peptides (e.g., a-defensins) into the mucus that control the concentration and adherence of bacteria to the surface epithelium. The mucus also contains secretory IgA, glycolytic and proteolytic enzymes that also help regulate the intestinal bacterial ecosystem and prevent bacterial penetration into the lamina propria [3]. Entry of bacteria and soluble antigens into the mucosa is normally very selective and carefully controlled. Specialized areas of the mucosa contain gut-associated lymphoid tissue (GALT) overlaid by modified intestinal epithelial cells called microfold (M) cells. The GALT holds the largest concentration of lymphocytes in the body. M cells capture, transport macromolecules and transfer them over to GALT dendritic cells. These cells then migrate to mesenteric lymph nodes that drain lymph from the intestine, and process and present antigen to naı¨ve T cells. The complex luminal environment of the intestine is also sampled directly by intraepithelial projections of lamina propria dendritic cells. These cells interact with intestinal immune cells, which eliminate threats or become tolerant to these antigens. This cross-talk involves the generation of T regulatory cells (Treg), which dampen the inflammatory response. Resident macrophages act like scavengers to remove bacteria and debris, remarkably without activating an immune response. B cells undergo an isotype switch and secrete IgA into the lumen. In summary, physical confinement of the intestinal microbiota to the intestinal lumen, limitation of activation of innate immune cells, induction of adaptive immune tolerance to commensal microbes, and direct inhibition of effector T-cell differentiation and function ensure a mutually beneficial relationship between the host and the gut microbiota under normal or steady-state conditions.

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For reasons that are not well understood, this delicate balance is perturbed in inflammatory bowel diseases (IBD). The primary events that lead to IBD involve a breakdown in the epithelial cell barrier, loss of tolerance to commensal intestinal bacteria, dysfunction of the innate immune system, and hyperactivation of the adaptive immune system. Patients with Crohn’s disease and their healthy first-degree relatives appear to have a primary increase in mucosal permeability [4, 5], which may increase exposure to luminal antigens in the lamina propria. Recently, it has been proposed that defective macrophage function may be a basic pathogenic mechanism for IBD. In this model, large numbers of microorganisms that gain access to the intestinal mucosa are not effectively cleared, triggering secondary activation of T cells, tissue damage, and clinical symptoms [6]. This hypothesis is plausible, since primary defects in phagocytes such as chronic granulomatous disease, Hermansky-Pudlak syndrome, and glycogen storage disease-Ib can present with IBD-like features [7–9]. Secondary T-cell activation is largely responsible for IBD chronicity, as discussed in the next section.

Innate immune functions and IBD pathogenesis As mentioned, there is mounting evidence that defects in the innate immune system are critical to the development of IBD. The innate system is constituted by multiple cell types (dendritic cells, macrophages, neutrophils, eosinophils, natural killer T cells, and gd T cells). These cells have receptors called pattern recognition receptors (PRRs) that detect molecules common to multiple microorganisms, termed pathogen-associated molecular patterns (PAMPs) [10]. These invariable and predetermined receptors are encoded in the germ-line. PAMPs are recognized by PRRs such as Toll-like receptors (TLRs). Unlike the state of “controlled inflammation” described above, where macrophages clear microbes without activating inflammation, in the context of IBD activated macrophages express TLRs [11, 12]. Their recognition and binding to PAMPs stimulates the production of cytokines that amplifies the immune response leading to the activation of the adaptive immune system. Nucleotide-binding oligomerization domain (NOD)-2, which recognizes bacterial muramyl dipeptide inside of the cells [13], is defective in 20% of Caucasians with Crohn’s disease. Loss-of-function mutations associated with three NOD2 polymorphisms are associated with stricturing and penetrating Crohn’s disease of the distal ileum [14, 15]. Intestinal dendritic cells directly sample the luminal environment of the intestine, or receive antigen from M cells. Under normal circumstances, they process and present these antigens via MHC to T cells in the draining mesenteric lymph nodes. These intestinal dendritic cells express low levels of co-stimulators and cytokines, typically resulting in T-cell tolerance. In response, T cells produce cytokines (IL-4, IL-10, TGF-b) that promote the differentiation of Tregs, which dampen inflammation. However, in IBD dendritic cells, which are activated by PAMPs that migrate to the GALT, express high levels of cytokines and co-stimulatory molecules. These, in turn, induce effector T-cell differentiation [16]. Importantly, they stimulate expression of a4b7, a gut-homing integrin in T cells in a process that involves retinoic acid [17]. As a result, activated T cells

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can reach the circulation and then return to the gut. A portion of these cells can migrate and stay in the bone marrow, and become activated by circulating dendritic cells, loaded with bacterial antigens [18]. This local immune response may have important implications for the function of bone cells in patients with IBD. Although intestinal epithelial cells are not considered part of the classic innate immune system, they are also immunocompetent. They express TLR2, 4, 9 [19] and NOD1 and 2 [20, 21], so they can directly sense intestinal bacteria. These interactions strengthen the epithelial cell barrier and protect against the onset of colitis.

The adaptive immune system and IBD T cells represent the adaptive immune system in the intestine. They reside in the lamina propria and in the intraepithelial compartment. T cells that express the ab T-cell receptor (TCR) reside in the lamina propria, whereas gd TCR cells can be found in the intraepithelial space. CD4þ ab T cells are critical to the pathogenesis of IBD. In mesenteric lymph nodes ab TCRs recognize cognate antigens presented by dendritic cells that have migrated from the intestinal mucosa after bacterial invasion or sampling of the luminal content. This constitutes signal 1 for T-cell activation, but a second signal is needed to fully activate the cell. Signal 2 is also present in the membrane of the antigen-presenting cell in the form of CD80 (B7.1) or CD86 (B7.2), which bind to CD28 on the surface of the T cell. However, binding of CD80/CD86 to CTLA-4 on the T-cell membrane inhibits T-cell activation. In addition, antigen binding to the TCR without co-stimulation typically renders the cell incapable of reacting to the antigen upon future re-exposure (a state called anergy) [2]. Activation of T cells in a defined cytokine context in the mesenteric lymph node directs them to a specific effector T helper (Th) phenotype. In the presence of IL-12 and IFN (interferon)-g, T cells sequentially activate STAT (Signal Transducers and Activators of Transcription)-1 and STAT-4, acquire the ability to secrete IFN-g, and are referred to as Th1 cells, which control intracellular pathogens through activation of innate cells like macrophages. IL-27 may also play a role in the differentiation of Th1 cells, especially in the intestine. The signature transcription factor for Th1 cells is T-bet (T-box transcription factor expressed in T cells). Th1 cells characteristically express CCR1, CCR5, and CXCR3 on their surface. On the other hand, Th2 cells are generated in the presence of IL-4, which activates STAT-6 and expression of the transcription factor GATA3. Th2 cells can contribute to allergic responses and help combat parasites through the synthesis of IL-4, IL-5, and IL-13. They can be recognized by their expression of the surface receptors CCR3 and CCR4. IL-6 and TGF-b direct cells toward a Th17 phenotype, which fight off fungi and extracellular bacteria. Th17 cells secrete IL-17, IL-21, IL-22, recruit neutrophils to the site of inflammation, and enhance epithelial barrier function. They are characterized by expression of the transcription factor ROR (retinoic acid receptor-related orphan nuclear receptor)-gt and the surface markers CCR4, CCR6, and IL-23R. IL-23 is a key cytokine in mediating IBD, which sustains a pathogenic pool of Th17 cells [22, 23]. Polymorphisms of

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the IL23R confer various levels of susceptibility to IBD [24]. IL-21 is also regarded as an important factor in human IBD perhaps by deregulating tolerance to microbial gut flora [25]. A fourth Th subpopulation called T follicular helper cells – T(FH) – secrete IL-21, express CXCR5 and Bcl-6 [26], and migrate into B-cell follicles where they provide support for B-cell function. They are also detected in gut-associated Peyer’s patches in a CD155-dependent manner [27]. Although Th1 and Th17 cells are present in small numbers in the non-inflamed lamina propria, CD4þ T cells that produce IL-10 and Treg are abundant [28]. These cells suppress the intestinal immune response despite permanent exposure to an enormous load of foreign antigens. These T-cell phenotypes are not fixed, and trans-differentiation may occur in response to changes in luminal environmental conditions [29, 30]. For example, Tregs have been shown to differentiate into T(FH) cells in Peyer’s patches [27]. In addition, “dual expressors” of IFN-g and Th17 have been reported [31]. Once activated, these effector T cells leave the mesenteric lymph nodes, enter the circulation, and can home to sites containing their cognate antigens. T cells activated by intestinal dendritic cells are imprinted to return to their original inductive site. They acquire mucosal addressins, including integrins and chemokine receptors, that enable them to home back to the intestine. For example, the integrin a4b7 binds to MAdCAM (Mucosal Addressin Cellular Adhesion Molecule)-1 and a4 to VCAM (Vascular Cell Adhesion Molecule)-1 in the vascular endothelium of specialized lamina propria postcapillary venules. The chemokine receptor CCR9 attaches to small intestinal epithelium CCL25 [32], CCR10 to colonic epithelial CCL28 [33], and CCR6 to CCL20 in small intestinal Peyer’s patches [34]. These and other receptors are upregulated during intestinal inflammation, promoting cellular infiltration of specific mucosal sites. Activated T cells then secrete cytokines that increase intestinal and vascular permeability, blood flow, the influx of inflammatory cells, and the production of oxygen radicals, matrix metalloproteinases, and other enzymes, resulting in tissue damage and symptoms of IBD. T cells express TLRs and can directly recognize PAMPs. There is experimental evidence that this can result in T-cell proliferation and production of pro-inflammatory factors in the intestinal lamina propria without the need for antigen presentation by dendritic cells or macrophages. This may be another mechanism by which microorganisms can regulate inflammation [35].

Immunological differences between Crohn’s disease and ulcerative colitis Until recently, Crohn’s disease was considered a Th1-mediated chronic inflammatory disease. This was based on the presence of CD4þ T cells expressing T-bet and IFN-g in the inflamed intestine of mice and humans [36], the ability of an IFN-g antibody to treat early IBD in mice, and the lack of colitis development in immunodeficient mice that received T-bet-deficient T cells. However, this paradigm was recently challenged by the observations that key molecules associated with the development, function, and

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maintenance of Th17 cells are abundant in the intestines of patients with Crohn’s disease. Th17 cells are more numerous in the inflamed colon of IL-10-null mice and mice adoptively transferred with CD45RBHi cells [22, 37]. Elson et al. have observed that Th17 cells, reactive to intestinal bacteria, produce colitis at much lower doses than Th1 cells in adoptive transfer models of colitis [23]. IL-23 is a critical cytokine in the perpetuation of the pathogenic Th17 cells pool, and plays other important Th17-independent roles in the pathogenesis of IBD. Genes involved in the IL-23/Th17 system such as IL-23R modulate susceptibility to IBD [38]. It is of interest that blockade of the common IL-12/IL-23 p40 subunit [39] and IL-23p19 ameliorate intestinal inflammation [23]. These hypotheses may not be mutually exclusive. It is possible that Th1 cells initiate the aberrant immune response, and that Th17 cells perpetuate it. Compared to Crohn’s disease, there is a paucity of data about the pathogenesis of ulcerative colitis, due to a relative lack of adequate experimental models. The inflammation in ulcerative colitis is considered a “modified Th2 response”, given the abundant presence of IL-13 and other Th2-type cytokines, like IL-5 [40]. NKT cells, which mature in the thymus in response to lipid antigen presentation by the Cd1d complex, are the main source of IL-13 in ulcerative colitis [41]. In the oxazolone mouse colitis model, which is Th2-dependent, IL-4 is important in the early phase of inflammation, whereas IL-13 drives chronic disease [41]. The IL-23/Th17 system appears to be important in the development of ulcerative colitis as well [38].

IBD and osteoimmunology As mentioned above, IBD is characterized by a vigorous, chronic immune response to luminal microbial antigens. This response is produced by activated T and B cells, and results in the production of a variety of pro-inflammatory factors that damage the intestinal lining. It is well documented that both children and adults with IBD frequently have bone mass deficits, even before treatment is started. In this section we will describe the impact of IBD on bone health and possible inflammatory mechanisms by which IBD affects bone mass.

Effects of IBD on bone mass IBD can be diagnosed at any age, but in children it is most commonly diagnosed during adolescence, a time normally characterized by rapid linear growth and bone mineral accrual. Crohn’s disease in particular is frequently associated with growth stunting and pubertal delay, which can affect the acquisition of bone mass. Laboratory, clinical, and bone biopsy evidence suggests that bone formation is markedly decreased in children with Crohn’s disease at diagnosis, before anti-inflammatory treatment is instituted, while bone resorption is also reduced [1, 42, 43]. The second peak in incidence of IBD is in young adults in the fourth decade of life. In these patients there is bone biomarker evidence of decreased bone formation and increased bone resorption [44–46]. Therefore,

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intestinal inflammation appears to primarily suppress bone formation, with uncoupling of bone remodeling in adults. The prevalence of bone mass deficits varies widely in IBD, depending on the population studied and how bone mass was measured [47]. The frequency of severe bone demineralization is ~10–20% in adults and children. Disease-related factors that are independent of treatment probably play an important role, since bone mass deficits are present from the time of diagnosis [1, 48]. In children with newly diagnosed Crohn’s disease, the tibia exhibits decreased trabecular bone mineral density, expanded endocortical surface and reduced periosteal circumference, resulting in a mechanically weaker bone [48]. However, it is not clear whether these biomechanical alterations result in an increased risk of fractures in patients with IBD. Retrospective population-based cohort studies of fractures requiring hospital care in adults with IBD range from no increase to moderate increase compared to the general population [49–54]. However, these studies did not account for clinically silent vertebral fractures. Case series suggest that these may be present in up to 1:5 adults with IBD [55, 56], even in patients with normal BMD [57]. Vertebral fractures have also been reported in children with Crohn’s disease [58]. More studies are needed to define the true risk of these fractures in adults and children.

Mechanisms by which IBD affects bone mass General disease-related and treatment factors The pathogenesis of bone loss in IBD is complex and likely involves disease and treatment factors. IBD, especially Crohn’s disease, is commonly associated with decreased calorieprotein and micronutrient intake, which can adversely affect bone accrual and maintenance [59, 60]. Vitamin D deficiency is prevalent in patients with IBD, especially in northern latitudes [61], which may impair the intestinal absorption of calcium, and in the rodent model of colitis exacerbate inflammation [62]. Vitamin K, a co-factor in the g-carboxylation of the glutamic acid of osteocalcin, may be deficient in IBD as well, affecting bone mineralization [63, 64]. Therefore, malnutrition may impair skeletal health in IBD. Sex steroids and the growth hormone/insulin-like growth factor-1 (IGF-1) axis are frequently disturbed in active IBD. Delayed puberty is common in adolescent children with Crohn’s disease and may affect the acquisition of peak bone mass [65]. Hypogonadism can be seen in adults and may impair bone remodeling [66]. Malnutrition and a decrease in growth hormone signaling in the liver and peripheral tissues act synergistically to decrease serum and tissue IGF-1 in children with active intestinal inflammation [67]. Low IGF-1 may impair longitudinal growth by chondrocytes, expansion of the outer cortical layer by periosteal osteoblasts, and recruitment of undifferentiated stromal cells into the osteoblast lineage [68, 69]. Large mechanical forces from skeletal muscles are important for the normal development and maintenance of bone mass [70]. Significant deficits in lean body mass,

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a surrogate for muscle mass, are prevalent in newly diagnosed children with Crohn’s disease and persist despite clinical improvement [71, 72]. Sarcopenia may be a consequence of inflammation, malnutrition, glucocorticoid use, and decreased physical activity. Medications to treat IBD can also impact bone cell function. Glucocorticoids (GC) are commonly used to induce remission in IBD. GC can inhibit bone formation, secondarily increase bone resorption, decrease muscle mass, and can increase fracture risk even at small doses [54, 73]. However, by decreasing inflammation, increasing appetite and weight gain and general well-being, GC may have positive effects on bone health as well. For this reason the role of GC in inducing bone deficits in IBD remains controversial. In addition, it is difficult to separate the effects of the underlying IBD from those of GC in the individual patient [74]. Infliximab, a TNF-a-specific antibody that is used to treat refractory IBD, improves biomarkers of bone formation in adults [75] and children [76], suggesting that adequate control of inflammation improves bone formation. Calcineurin inhibitors are used primarily in hospitalized patients with ulcerative colitis unresponsive to intravenous GC. Calcineurin is a calcium-binding phosphatase that regulates osteoblast and osteoclast function [77]. A substrate of calcineurin, nuclear factor of activated T cells (NFAT), is critically important in osteoblast and osteoclast differentiation [78]. Therefore, calcineurin inhibitors may modulate the development of osteoblasts and osteoclasts in patients with IBD.

Disease-related immune factors As described above, the intestine in IBD harbors activated epithelial cells, dendritic cells, macrophages, and T and B cells that secrete a vast array of inflammatory factors. These can spill over into the circulation and affect bone cell function (Figure 11-1). For example, it is well documented that serum IL-6 is elevated in active IBD [79, 80]. Serum from newly diagnosed children with Crohn’s disease decreases bone formation in bone explants [81]. Antibody neutralization of serum IL-6 reverses the effects of serum in this model [43], suggesting that IL-6 decreases bone remodeling in IBD. TNF-a is a central cytokine in the pathogenesis of IBD. Mice injected with TNF-a and mice with TNBS (trinitrobenzene sulfonic acid)-induced colitis, have decreased expression of osteoblast genes involved in mineralization. Systemic administration of anti-TNF-a antibodies rescues this expression [82]. In humans, TNF-a-specific antibodies produce a significant increase in serum biomarkers of bone formation, especially in children with severe IBD [76]. Other proinflammatory factors produced by the inflamed intestine, such as IL-23 and IFN-g, may also modulate bone cell function. Importantly, it is not clear whether the beneficial effects of TNF-a neutralization on bone are secondary to a non-specific decrease in intestinal inflammation, to direct effects in the bone micro-environment, or both. Experimental evidence suggests that bone marrow cells become activated in the presence of intestinal inflammation and secrete TNF-a [83]. Although the mechanisms by which this happens are not yet understood,

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DC = Dendritic cells aDC= Activated DC CM = Central memory T cells EM = Effector memory T cells PAMPs = Pathogen-associated molecular patterns

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Autophagy defects? Microbial recognition issues?

FIGURE 11-1 (A) The intestine is colonized with over 100 trillion microorganisms, which are contained in its lumen by a single layer of epithelial cells. In the steady state, the host tolerates this enormous antigenic load due to special adaptations of dendritic cells (DC), macrophages, and the presence of regulatory T cells. In IBD, these immune-suppressive mechanisms are lost, and dendritic cells, macrophages, and T cells become activated. T cells acquire immunological memory for their cognate antigen (central memory and effector memory cells), and intestinal mucosal addressins. T memory cells and dendritic cells leave mesenteric lymph nodes, Peyer’s patches and intestinal lymphoid follicles, enter the circulation and home back to the intestine or establish residence in the bone marrow. In addition, pro-inflammatory factors, endotoxin, flagellin, and other bacterial products may enter the circulation and gain access to the bone microenvironment. (B) In bone, dendritic cells that carry antigen may activate the differentiation of T cells into effector cell lineages. The commitment to Th1, Th17, or Th2 cells will depend on the cytokine milieu. In addition, microbial antigens may be taken up by resident bone cells like osteomacs and activate immune responses that affect bone cell function. IBD is associated with innate immune defects that may facilitate the survival of gut-derived microorganisms, and generate vigorous adaptive immune response. Please refer to color plate section.

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there are several possibilities. The bone marrow contains central memory CD8þ T cells that are capable of responding to antigen [84]. Circulating dendritic cells originating from the inflamed gut and loaded with antigen may encounter these cells and activate them [18, 85, 86]. The bone marrow can be the site where T cells first encounter antigen [87], so it is possible that PAMPs and other microbial antigens leaked by the intestine into the systemic circulation reach the bone marrow and after processing turn these cells on. Another subset of cells in the bone marrow may also be capable of cytokine secretion after antigen stimulation. This is a pool of effector memory CD4þ cells that originates in the inflamed gut and is maintained in the bone marrow by IL-7-producing cells [88]. These cells are capable of “transmitting” colitis when injected into an immunodeficient mouse recipient. Ex vivo activation results in high levels of TNF-a and IFN-g. Therefore, several possible mechanisms exist that may facilitate the local production of cytokines in the bone microenvironment, affecting bone cell function. Another possibility is that resident dendritic cells differentiate into functional, resorbing osteoclasts in bone under inflammatory conditions. The lack of skeletal defects in dendritic-cell-deficient mice suggests that they probably do not play a role in bone remodeling under homeostatic conditions [89]. However, in the bone environment immune interactions between immature dendritic cells, CD4þ T cells and microbial products, which may occur in IBD can stimulate their development into functional osteoclasts in mice [90, 91]. Autophagy is a process by which cells degrade defective organelles, proteins, and bacteria. Defects in the autophagy-related genes ATG16L1 and IRGM are associated with susceptibility to Crohn’s disease, but not ulcerative colitis [92–94]. It is unclear whether alterations in autophagy play any role in bone physiology in Crohn’s disease. However, it is theoretically possible that osteoclasts, which are related to phagocytic cells with normally active autophagy, may be affected; but this remains to be tested. Recently discovered macrophage-like cells (osteomacs), which overlay osteoblasts in endosteal and periosteal surfaces may also be a target of inflammatory responses. These cells may be activated by circulating microbial antigens or by neighboring immune cells and influence bone formative activity [95]. Finally, bone cells themselves may act as immunocompetent partners. Both osteoblasts and osteoclasts are capable of secreting pro-inflammatory factors when stimulated by bacterial products and cytokines [96, 97]. They express Toll-like and NOD-like receptors upon exposure to PAMPs, and therefore can directly sense microbial-derived molecules [98–100]. In consequence, translocation of microbial products into the circulation of patients with IBD may affect the function of these cells directly.

RANKL and OPG RANKL (receptor activator of nuclear factor-kB-ligand) is essential for normal osteoclast formation. RANKL binds to its receptor RANK on osteoclast precursors and stimulates their differentiation and activity. Osteoprotegerin (OPG) is a soluble decoy receptor for

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RANKL [101]. Additionally, RANKL has well-defined immunologic effects, described in detail elsewhere in this textbook [102]. The discovery of RANKL and the fact that it is produced by activated T cells [103] sparked a search for a role in the pathogenesis of bone loss in IBD. Ashcroft et al. reported in the IL-2 null mouse model of inflammation that RANKL increased the survival of intestinal dendritic cells. Furthermore, exogenous OPG decreased the number of these cells, ameliorated the severity of colitis, and improved bone density [85]. Although OPG also improved bone mass in an adoptive transfer model of colitis, it had no effect on intestinal inflammation [83]. Clinical studies performed in adult patients reveal that the expression of OPG and RANKL is upregulated in the inflamed intestine [104], and that the expression of OPG predicts response to antiTNF-a therapy [105]. OPG is constitutively expressed by intestinal epithelial cells, and TNF-a increases its production [106]. OPG is also expressed by macrophages and dendritic cells in patients with IBD [107, 108], suggesting that it plays an immunomodulatory role. This function may be a consequence of OPG binding to the pro-apoptic molecule TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) [109]. IBD is characterized by decreased apoptosis of activated immune cells, and it is possible that OPG prolongs the survival of these cells by neutralizing TRAIL [110]. OPG is modestly elevated in serum from patients with IBD [111]. It has been suggested that this represents either a physiologic response to increased bone resorption or is a marker of active inflammation [112, 113].

Conclusion IBD is a complex clinical disorder, with various clinical phenotypes. It is likely that its effects on bone are mediated at least in part by inflammation. The skeleton may not be just a passive recipient of gut-derived inflammatory factors and cells, but an active participant. Thus immune reaction in bone may involve activated cells that originate in the intestine, and/or resident cells. In addition, primary defects in innate immunity and autophagy may affect the function of osteoblasts and osteoclasts. Much remains to be understood about these mechanisms, and the clinical consequences of bone mass deficits, including fractures.

References [1] Sylvester FA, Wyzga N, Hyams JS, Davis PM, Lerer T, Vance K, Hawker G, Griffiths AM. Natural history of bone metabolism and bone mineral density in children with inflammatory bowel disease. Inflamm Bowel Dis 2007;13:42–50. [2] Maynard CL, Weaver CT. Intestinal effector T cells in health and disease. Immunity 2009;31: 389–400. [3] Nieuwenhuis EE, Blumberg RS. The role of the epithelial barrier in inflammatory bowel disease. Adv Exp Med Biol 2006;579:108–16. [4] Benjamin J, Makharia GK, Joshi YK. Association between intestinal permeability and anti-Saccharomyces cerevisiae antibodies in patients with Crohn’s disease. Inflamm Bowel Dis 2008;14:1610–11.

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