Original Article

Toxicologic Pathology
1-14

Bone and the Immune System ª The Author(s) 2017

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DOI: 10.1177/0192623317735316
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M. Neale Weitzmann1,2

Abstract
Osteoporosis increases fracture risk, a cause of crippling morbidity and mortality. The immunoskeletal interface (ISI) is a cen-
tralization of cell and cytokine effectors shared between skeletal and immune systems. Consequently, the immune system mediates
powerful effects on bone turnover. Physiologically, B cells secrete osteoprotegerin (OPG), a potent anti-osteoclastogenic factor
that preserves bone mass. However, activated T cells and B cells secrete pro-osteoclastogenic factors including receptor activator
of Nuclear factor-kappaB (NF-kB) ligand (RANKL), Interleukin (IL)-17A, and tumor necrosis factor (TNF)-a promoting bone loss in
inflammatory states such as rheumatoid arthritis. Recently, ISI disruption has been linked to osteoporosis in human immunode-
ficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS), where elevated B cell RANKL and diminished OPG
drive bone resorption. HIV-antiretroviral therapy paradoxically intensifies bone loss during disease reversal, as immune recon-
stitution produces osteoclastogenic cytokines. Interestingly, in estrogen deficiency, activated T cells secrete RANKL, TNF, and IL-
17A that amplify bone resorption and contribute to postmenopausal osteoporosis. T cell–produced TNF and IL-17A further
contribute to bone loss in hyperparathyroidism, while T cell production of the anabolic Wingless integration site (Wnt) ligand,
Wnt10b, promotes bone formation in response to anabolic parathyroid hormone and the immunomodulatory costimulation
inhibitor cytotoxic T lymphocyte–associated protein-4-IgG (abatacept). These findings provide a window into the workings of the
ISI and suggest novel targets for future therapeutic interventions to reduce fracture risk.

Keywords
bone, immunoskeletal interface, T cell, B cell, RANKL, osteoprotegerin, osteoimmunology

Osteoporosis (Latin for porous bone) is a common malady of
the skeleton and ensues when the rate of osteoclastic bone
resorption outpaces the rate of osteoblastic bone formation,
conditions that predispose to bone loss. From a clinical perspec-
tive, osteoporosis is most commonly defined as a bone mineral
density (BMD) T score of less than or equal to 2.5 standard
deviations from a young adult reference range, following a bone
densitometry scan using dual-energy X-ray absorptiometry
(DXA; Unnanuntana et al. 2010). From a biological standpoint
and at the molecular level, osteoporotic bone is characterized by
a loss of BMD and structural integrity, leading to a conforma-
tion with diminished load-bearing capacity and with an
increased propensity to fracture.
The consequences of fragility fractures can be devastating
and are commonly associated with crippling disability that
necessitates intensive rehabilitation in as many as 75% of
cases. In the case of hip fractures, mortality rates of 24% to
33% are common in the first year following a fracture (Bass
et al. 2007; Johnell and Kanis 2006; Lewis et al. 2006). The
associated loss of independence during and often beyond, the
fracture rehabilitation period, can lead to a marked decline in
quality of life especially in aged fracture victims (Kates, Kates,
and Mendelson 2007; Johnell and Kanis 2006). It is estimated
that almost 50% of women and 30% of men over the age of 50
will suffer a fragility fracture due to osteoporosis (Eisman et al.
2012), and the worldwide incidence of fracture is expected to
rise to 6 million by 2050 (Cummings and Melton 2002) with
costs projected to reach US$25 billion in the United States
alone by 2025 (Burge et al. 2007).
An interesting and unexpected finding that has emerged in
the field of bone biology is the repurposing by the skeleton of
key immune elements including adaptive and innate immune
cells and key immune-derived cytokines. As a result, physio-
logical functions of the adaptive immune system beneficially
regulate the skeleton. However, under states of pathologic
immune dysfunction such as immunodeficiency or inflamma-
tory responses to infection/disease, the skeleton is at the mercy
of the immune response and serious collateral damage to bone
may result leading to osteoporosis and an elevated risk for bone
1
Department of Veterans Affairs, Atlanta VA Medical Center, Decatur,
Georgia, USA
2
Department of Medicine, Division of Endocrinology and Metabolism and
Lipids, Emory University School of Medicine, Atlanta, Georgia, USA
Corresponding Author:
M. Neale Weitzmann, Department of Medicine, Division of Endocrinology and
Metabolism and Lipids, Emory University School of Medicine, 101 Woodruff
Circle, 1305 WMB, Atlanta, GA 30322, USA.
Email: mweitzm@emory.edu
2 Toxicologic Pathology XX(X)
fracture (Weitzmann and Ofotokun 2016; Weitzmann and
Pacifici 2006). Although bone loss is a natural consequence
of aging, it is exacerbated by many common pathological
conditions that afflict many people. This includes inflamma-
tory diseases such as rheumatoid arthritis (RA), periodontal
infection, and inflammatory bowel diseases including Crones
disease. The immunodeficiency state resulting from HIV infec-
tion further promotes bone resorption. Another key protagonist
of exacerbated skeletal decline is the result of estrogen decline
following the menopause (postmenopausal osteoporosis).
These bone loss states are all characterized by an increase in
osteoclastic bone resorption relative to osteoblastic bone for-
mation, leading to net bone loss (Weitzmann and Ofotokun
2016; Weitzmann and Pacifici 2006).
Although it has long been recognized that osteoclast precur-
sors derive from the monocyte/macrophage lineage (Walker
1972, 1975; Buring 1975), cells of immune origin, in the past
2 decades, the new field of “osteoimmunology” has blossomed,
revealing a deeply integrated immunoskeletal interface (ISI) in
which immune cells direct physiological and pathological bone
resorption and bone formation (Weitzmann and Ofotokun
2016; Weitzmann and Pacifici 2006).
Although many of the concepts described below have been
uncovered using animal models, predominantly mice and rats,
tantalizing evidence is beginning to emerge to support a role
for the immune response in human bone diseases. It should be
pointed out, however, that osteoimmunology does not replace
traditional bone biology. In most cases, the ISI simply adds a
new layer to the underlying principles already elucidated.
The following review outlines some of the past and ongoing
investigations by our group and others into the role of the ISI in
the regulation of the skeleton.
Osteoclasts and the Receptor Activator of
Nuclear factor-kappaB (NF-kB) (RANK)/
Receptor Activator of NF-kB Ligand
(RANKL)/Osteoprotegerin (OPG) System
Although osteoclasts are the sole bone resorbing cells of the
body and increased osteoclastic bone resorption underlies
bone loss and osteoporosis development, it is only compara-
tively recently that the process underlying osteoclastogenesis
has been unveiled. It has long been recognized that osteoclast
precursors are significantly more frequent in inflammatory
conditions in both humans and animals, particularly when
inflammatory cytokines are elevated (Pacifici et al. 1991;
Kitazawa et al. 1994; Kimble et al. 1996). However, adding
inflammatory cytokines to osteoclast precursors is under most
circumstances ineffective in promoting osteoclast formation
suggesting an alternative mechanism.
In 1997 and 1998, a series of key discoveries unveiled a
previously unknown pathway for stimulating osteoclast forma-
tion (Simonet et al. 1997). It is now evident that what differenti-
ates an osteoclast precursor from a regular monocyte is
expression on its surface of a specific molecule, RANK. RANK
is the receptor for the key osteoclastogenic cytokine RANK
ligand (RANKL; Simonet et al. 1997; Anderson et al. 1997;
Wong et al. 1997), which upon binding to RANK in the presence
of the trophic factor, macrophage colony-stimulating factor,
causes the osteoclast precursor to differentiate into a preosteo-
clast (Lacey et al. 1998). These preosteoclasts further fuse
together to form giant multinucleated bone resorbing mature
osteoclasts. As with many biological pathways, there are checks
and balances, and in this pathway, this takes the form of a soluble
RANKL decoy receptor called osteoprotegerin (OPG; Lacey
et al. 1998; Tsuda et al. 1997). OPG binds to RANKL and pre-
vents it from associating with RANK, thus reducing the rate of
osteoclast differentiation and bone resorption. As RANKL and
OPG are the final downstream effectors of osteoclastogenesis,
the ratio of RANKL to that of OPG in the bone marrow micro-
environment is a key determinant of the rate of osteoclastic bone
resorption occurring in humans and animals (Teitelbaum 2000).
This pathway is summarized diagrammatically in Figure 1.
It is further recognized that the role of inflammatory cytokines
in promoting osteoclastogenesis is indirect and mediated through
the regulation of the RANK/RANKL/OPG axis. For example,
tumor necrosis factor alpha (TNF), a potent inflammatory and
indeed osteoclastogenic cytokine, drives up bone resorption by
promoting RANK expression on monocytes thus converting them
into osteoclast precursors, upregulates RANKL expression by
osteoblast lineage cells (Hofbauer et al. 1999), and down mod-
ulates osteoblastic production of OPG. Although other inflamma-
tory cytokines such as IL-1, IL-6, IL-7, and Interleukin (IL)-17A
also upregulate osteoclastogenesis by regulating RANK,
RANKL, and/or OPG production, TNF mediates 1 other unique
function making it an extremely potent osteoclastogenic factor.
TNF synergizes with RANKL at the signal transduction level to
further intensify osteoclastogenesis and bone resorption (Cenci
et al. 2000; Lam et al. 2000; Zhang et al. 2001; Fuller et al. 2002).
The ISI
Although long recognized that osteoclasts derive from cells of the
monocyte lineage (immune cells), an unexpected finding was the
existence of a deeply rooted nexus between the immune and ske-
letal systems, the ISI. This is the result of a centralization of mul-
tiple cells of the adaptive immune response and their cytokine
effectors, which serve dual functions in the regulation of the ske-
leton. One key aspect of this involves activated lymphocytes, both
T- and B cells, which secrete RANKL and TNF under inflamma-
tory conditions, driving up osteoclast formation and bone resorp-
tion in inflammatory states including the autoimmune disease RA,
inflammatory bowel diseases including Crohn’s disease, and per-
iodontal infection. Consequently, these conditions are all associ-
ated with bone loss (Weitzmann and Ofotokun 2016).
In contrast to these inflammatory states, under physiological
conditions, B cells are a significant source of the osteoclast
inhibitor OPG. Early studies recognized that human tonsil B
cells secrete OPG and that in vitro activation of cluster of
differentiation (CD) 40 costimulatory pathway on B cells fur-
ther upregulates OPG production (Yun et al. 1998). We later
Weitzmann 3

Figure 1. Osteoclastogenesis and the receptor activator of NF-kB (RANK)/receptor activator of NF-kB ligand (RANKL)/osteoprotegerin (OPG)
axis. Osteoclast precursors derive from the monocyte lineage and express the RANK that is a receptor for the key osteoclastogenic cytokine
RANKL. RANKL, in the presence of permissive concentrations of the tropic factor, macrophage colony-stimulating factor (M-CSF), causes
differentiation of osteoclast precursors into preosteoclasts that fuse together to form mature multinucleated bone resorbing osteoclasts. RANKL
activity is moderated by a soluble decoy receptor OPG. Inflammatory cytokines promote osteoclastogenesis and bone resorption by stimulating
RANK and/or increasing production of RANKL and/or suppressing expression of OPG.

ratified these data in the murine system where we demonstrated
that in mouse bone marrow, the B cell lineage contributed 64%
of the total OPG production (Y. Li, Toraldo, et al. 2007). Con-
sistent with these data, B cell knockout (KO) mice were found
to have a bone marrow deficit in OPG, an enhanced rate of
osteoclastic bone resorption, and significantly diminished
BMD and bone mass. Reconstitution of B cells into young B
cell null mice rescued these alterations to the ISI and prevented
bone loss (Y. Li, Toraldo, et al. 2007).
As CD40 on B cells functions as a receptor for CD40 ligand
(CD40L) expressed predominantly by activated T cells, we fur-
ther investigated a role for T cell CD40/CD40L costimulation
and found that deletion of CD40 or CD40L or of T cells also led
to diminished B cell OPG production and bone loss (Y. Li,
Toraldo, et al. 2007).
Taken together, these data demonstrate that under inflam-
matory conditions B cells and T cells damage the skeleton
though copious secretion of RANKL and inflammatory cyto-
kines such as TNF. By contrast, under basal conditions, B cells,
regulated by costimulatory actions through T cells, protect the
skeleton by promoting production of OPG. This pathway is
summarized diagrammatically in Figure 2.
The Bone Loss of HIV-1 Infection
Given the existence of the ISI and the protective role of
immune cells on basal bone turnover, one might predict that
conditions that disrupt adaptive immunity would lead to an
imbalance in bone turnover promoting osteoclast formation,
increased bone resorption and bone loss. A test of this predic-
tion involves infection by the HIV-1 virus that devastates adap-
tive immunity leading to AIDS. HIV dramatically depletes
CD4þ T cells, the lynchpin of adaptive immunity, leading to
a decline in number and function of other adaptive immune
components including B cells and CD8þ T cells.
In fact, bone loss is widespread in human subjects infected
by HIV and a meta-analysis has concluded that overall there is
a 50% to 70% incidence of osteopenia and 15% incidence of
osteoporosis in this population (Brown and Qaqish 2006).
Importantly, this loss of BMD has been demonstrated in several
large population-based studies to translate into a significantly
elevated prevalence of bone fracture (Prior et al. 2007; Triant
et al. 2008; Young et al. 2011; Womack et al. 2011; Guerri-
Fernandez et al. 2013; Prieto-Alhambra et al. 2014; Sharma
et al. 2015). In fact, both men and women are affected and with
fracture prevalence affected over a wide age range, such that in
the context of HIV, even relatively younger men are at
increased risk of a fragility fracture (Triant et al. 2008).
The mechanisms underlying bone loss and increased fracture
incidence have been difficult to assess in humans, given a sig-
nificant number of coexisting osteoporosis risk factors in this
demographic. These include metabolic complications of AIDS
such as hypogonadism and renal disease and lifestyle factors
including high rates of smoking and alcohol and recreational
4 Toxicologic Pathology XX(X)

Figure 2. The immunoskeletal interface (ISI). The ISI is a nexus between immune and skeletal systems with key immune components repurposed
for skeletal functions. Under basal physiological conditions, B cell, under the regulation of T cells, secretes copious concentrations of the receptor
activator of NF-kB ligand (RANKL) decoy receptor osteoprotegerin (OPG), moderating osteoclastogenesis and maintaining homeostatic bone
turnover. Under pathological conditions such as inflammation, activated B cells and T cells secrete large concentrations of RANKL, driving up
osteoclastogenesis, disrupting basal bone homeostasis, and leading to bone loss.

drug use, which can all impact the skeleton. Finally, complex
invasive mechanistic studies in humans are severely limited in
scope, ultimately necessitating the use of an animal model. The
animal model we chose for these studies was the HIV transgenic
(Tg) rat, a small animal model of HIV infection that develops
multiple clinical manifestations of human AIDS (Reid et al.
2001). To interrogate changes in the skeleton, we performed
bone densitometry to quantify BMD using DXA. Tg rats dis-
played significantly diminished BMD at lumbar vertebrae and
femurs and in both trabecular and cortical compartments as
assessed by microcomputed tomography (μCT; Vikulina et al.
2010). Biochemical markers of in vivo bone resorption further
identified a significant increase in the bone resorption marker C-
terminal telopeptide of type I collagen (CTx), a sensitive and
specific marker of bone resorption, while osteocalcin, a marker
of bone formation, was unchanged. Histological and histomor-
phometric analysis of tartrate resistant acid phosphatase positive
multinucleated cells (osteoclasts) confirmed a significant
increase in osteoclast number and the area of bone surface cov-
ered by osteoclasts in HIV Tg rats. To understand the mechan-
istic basis for increased osteoclasts, we next examined the
expression of RANKL and OPG in the bone marrow by real-
time reverse transcriptase (RT)-polymerase chain reaction
(PCR) analysis of whole bone marrow and observed a significant
elevation in expression of RANKL and a simultaneous signifi-
cant decline in OPG. These data suggest an imbalance in the
RANKL/OPG ratio may underlie the osteoclastic bone loss in
HIV Tg rats. As many cell types have the capacity to secrete
both OPG and RANKL, we next investigated production of these
factors by specific cell populations. Immunomagnetic isolation
of B cells followed by RT-PCR confirmed that the changes in
RANKL and OPG observed were exclusively a consequence of
altered B cell function (Vikulina et al. 2010).
To determine whether these changes in B cell production of
RANKL and OPG are representative of human HIV, we per-
formed a translational clinical study in which we recruited 58
HIV-negative control subjects and 62 HIV-positive patients
and collected peripheral blood mononuclear cells and
plasma/serum (Titanji et al. 2014). As expected, we identified
a significant elevation in bone resorption marker (CTx) in HIV-
infected subjects compared to uninfected controls. Flow cyto-
metric analysis of peripheral blood B cells further ratified a
significant 20% decline in the percentage of OPG producing
B cells and a significant 60% increase in the percentage of B
cells expressing RANKL in conditions of HIV infection.
Although cells in the peripheral circulation may not accurately
represent conditions in the bone microenvironment and may
only provide muted evidence of changes at other sites, overall
the data strongly support an altered B cell RANKL/OPG ratio
in HIV-infected patients. Importantly, we further identified a
significant inverse correlation between BMD at the femoral
neck and total hip with the B cell RANKL/OPG ratio, suggest-
ing a pathophysiological effect of B cells on BMD (Titanji
et al. 2014).
Weitzmann 5

Figure 3. The bone loss of human immunodeficiency virus (HIV) infection. HIV infection leads to a disruption of the immunoskeletal interface.
Damage to adaptive immune components, especially CD4þ T cells, leads to a loss of costimualtion and changes in cytokine production, causing a
significant decline in B cell osteoprotegerin (OPG) production and a significant upswing in production of receptor activator of NF-kB ligand
(RANKL). This RANKL/OPG imbalance drives up osteoclastogenesis and bone resorption, leading to bone loss.

Although bone loss in HIV-infected subjects is likely multi-
factorial and exacerbated by AIDS sequelae and lifestyle fac-
tors, we propose that 1 major underlying cause is an
immunoskeletal defect in B cell RANKL and OPG production.
HIV infection likely results in significant alterations in CD4þ T
cell costimulation, a consequence of severe CD4 depletion as
well as functional changes. Irrespective of the underlying dis-
turbance, B cell function changes from that of OPG production
to that of RANKL production (an activated condition). The
increase in free bioactive RANKL thus increases osteoclast
differentiation, driving up bone resorption and leading to bone
loss. This model is presented diagrammatically in Figure 3.
The Bone Loss of HIV Antiretroviral
Therapy (ART)
Years (often decades) of undiagnosed HIV infection drive a
significant deterioration of the skeleton. Once diagnosed, how-
ever, the majority of patients begin ART also known as highly
active antiretroviral therapy and combinatorial antiretroviral
therapy. In most cases, ART comprises a cocktail of multiple
agents from different drug classes. Modern ART drug combi-
nations are extremely effective at reducing HIV viral load and
reversing many of the classic manifestations of AIDS.
Paradoxically, the skeleton does not improve with ART and
in fact, in most cases, actually undergoes a further deterioration
with an additional average loss of up to 6% in BMD within the
first 1 to 2 years of therapy initiation (McComsey et al. 2010).
Although specific ART classes and indeed specific drugs
within those classes appear to mediate more dramatic bone loss
than others (particularly tenofovir disoproxil fumarate contain-
ing regimens; Wohl et al. 2016), it is now recognized that
virtually all ART mediate some degree of bone loss, and recent
evidence supports the contention that much, if not most, of the
bone loss is independent of the specific antiviral agents used
(Brown et al. 2009; Piso et al. 2011; Bruera et al. 2003).
How ART affects the skeleton is unclear and has traditionally
been considered to be a direct toxic effect on bone cells such as
osteoblasts increasing RANKL production. However, studies of
antiretroviral drugs in culture systems (Gibellini et al. 2010;
Grigsby, Pham, Gopalakrishnan, et al. 2010; Grigsby, Pham,
Mansky, et al. 2010) or in vivo in mice (Wang et al. 2004)
generally fail to recapitulate the effects on bone turnover that
are observed when administered to humans with HIV infection.
Furthermore, recent studies in which ART is used to treat human
hepatitis B virus infection cause little bone loss compared to
HIV-infected subjects (Chan et al. 2016). Taken together, the
data do not support a direct toxic action of ART on bone cells
and suggest a mechanism unique to HIV infection.
What all HIV agents have in common is that they suppress
viral replication and reduce viral load. As a consequence,
adaptive immune function undergoes, in part, reconstitution
and reactivation. The recovery of CD4þ T cells involves
homeostatic reconstitution, a process involving T cell prolif-
eration and expansion to fill available immunological space
(Ernst et al. 1999; Surh and Sprent 2000). Similar processes
are involved in CD8þ T cell and B cell recovery and are
driven in part through cytokine-mediated processes. Through
costimulatory interactions and cytokine production, CD4þ T
cell subsets further regulate other adaptive immune
6 Toxicologic Pathology XX(X)

Figure 4. The bone loss of human immunodeficiency virus (HIV) antiretroviral therapy (ART). HIV ART significantly diminishes viral load allowing
a partial CD4þ T cell recovery. CD4þ T cells undergo homeostatic expansion and repopulation of immunological niches. This process is
inflammatory and leads to the release of receptor activator of NF-kB ligand (RANKL) and tumor necrosis factor (TNF) driving up osteoclasto-
genesis. In addition, reactivation of adaptive immune function causes additional RANKL and/or TNF production by APC, CD8þ T cells, and B cells.
These high levels of osteoclastogenic cytokines promote elevated osteoclastogenesis and stimulate bone resorption, leading to additional bone
loss over and above that causes by HIV infection itself.

components including humoral immunity (B cells) and anti-
gen presenting cells including macrophages, dendritic cells,
and B cells. The regeneration and rekindling of adaptive
immunity thus has the potential to produce inflammatory
events that may have the capacity to drive osteoclastogenesis
and bone loss, as is characteristic of other inflammatory states
(Kotake et al. 2001; P. Li and Schwarz 2003).
To test this hypothesis, we mimicked the process of T cell
repopulation following ART in humans by reconstituting T
cells by syngeneic adoptive transfer into T cell deficient TCRb
KO mice and quantified bone turnover and structure over
3 months (Ofotokun et al. 2015). Using prospective DXA to
quantify BMD, we found a dramatic loss of bone mass in
reconstituted mice, as T cells homeostatically expanded and
engaged adaptive immunity. μCT further revealed significant
deterioration of both cortical and trabecular bone compart-
ments. A marker of bone resorption (CTx) revealed signifi-
cantly increased bone resorption and serum levels of RANKL
and TNF were found to be significantly elevated, similarly to
human HIV patients initiating ART (Ofotokun et al. 2016).
Using flow cytometry of whole bone marrow and/or spleen,
we further demonstrated that both CD4þ and CD8þ T cells
produced significantly increased levels of RANKL, while mul-
tiple adaptive immune components including CD4þ and CD8þ
T cells, B cells, and macrophages all produced significant lev-
els of TNF. Reconstitution of T cells from RANKL KO mice
led to significant protection from bone loss, suggesting a key
role of T cell RANKL while transplantation of TNF KO T cells
only partly diminished bone loss, consistent with TNF produc-
tion by not only T cells but also other adaptive immune cells
(Ofotokun et al. 2015).
Taken together, the data confirm that homeostatic repopula-
tion of T cells leads to an inflammatory environment capable of
driving significant bone resorption and loss of BMD and mass.
This model is presented diagrammatically in Figure 4.
Whether this mechanism is a significant contributor to bone
loss in humans initiating ART remains to be determined,
although in support of this mechanism, recent clinical data
by us (Ofotokun et al. 2016) and others (Grant et al. 2013)
reveal that subjects with low starting CD4þ T cell number and
those undergoing the most robust T cell reconstitution (Ofoto-
kun et al. 2016) undergo to most aggressive bone loss.
A translational clinical study to validate homeostatic recon-
stitution and immune activation as a key mechanism underlying
ART bone loss is presently in progress.
Estrogen Deficiency Bone Loss and the Role
of the ISI
Postmenopausal osteoporosis is the archetypal bone disease of
women and is the result of an imbalance in bone turnover as a
consequence of estrogen deficiency following the menopause
(Weitzmann and Pacifici 2006). As in other osteoporotic con-
ditions, estrogen deficiency leads to an enhanced rate of bone
resorption relative to bone formation, leading to net bone loss.
Ovariectomy in mice or rats is a traditional animal model of
Weitzmann
postmenopausal osteoporosis, as removal of the ovaries causes
a rapid drop in estrogen levels that leads to increased bone
resorption and significant cortical and trabecular bone loss
within a period of just 2 to 4 weeks (Weitzmann et al. 2002).
Estrogen is established to mediate potent anti-inflammatory
effects in the body, and loss of estrogen has been shown to
cause significant expansion of lymphocytes, both T cells
(Cenci et al. 2003) and B cells (Miyaura et al. 1997). To test
whether T cell expansion following estrogen decline promotes
bone loss as in other inflammatory conditions, we performed
ovariectomy in T cell deficient nude mice (Cenci et al. 2000).
While control Wild Type (WT) mice underwent significant loss
of BMD due to increased bone resorption, nude mice were
protected from osteoclastic bone loss. Although activated T
cells are a significant source of RANKL, this cytokine is not
typically observed to be increased in T cells under conditions of
estrogen deficiency in mice. By contrast, TNF is significantly
increased in the bone marrow, a consequence of T cells (Cenci
et al. 2000). Although TNF production per cell was not
increased, the expansion of T cells in bone marrow and other
peripheral sites leads to an overall increase in TNF. TNF has
indeed been shown to be significantly elevated in both human
peripheral blood cells in surgically ovariectomized women
(Pacifici et al. 1991) and in mice (Cenci et al. 2000). Attesting
to the importance of TNF in ovariectomy bone loss, TNF and
TNF receptor I (p55) KO mice fail to undergo bone loss in
response to ovariectomy (Cenci et al. 2000). These studies led
to a model whereby estrogen decline leads to an expansion in T
cells secreting TNF, and this TNF amplifies RANKL-induced
osteoclastic bone resorption causing bone loss (Weitzmann and
Pacifici 2006). Although this general model still appears to
hold true, the regulation of the signaling cascades that lead to
T cell expansion has turned out to be exceedingly complex.
Among the key upstream events, driving T cell expansion is an
increase in IL-7 production by multiple tissues within the body
(Ryan et al. 2005). IL-7 is a potent lymphopoietic cytokine that
controls multiple steps in T cell biology. IL-7 increases the
sensitivity of T cells to otherwise tolerogenic antigens decreas-
ing the threshold for antigen-dependent T cell activation
(Weitzmann and Pacifici 2006). Differentiation of T cells into
different T-helper subsets including Th1 cells leads to produc-
tion of TNF but also of Interferon (IFN)g, a cytokine that
upregulates the transcription factor transactivator (CIITA) in
macrophages causing upregulation of major histocompatibility
complex II (MHCII) and increased antigen presentation to
T cells that further amplifies T cell activation (Cenci et al.
2003). Another helper subset induced by this process is
Th17, characterized by production of IL-17A, a potent osteo-
clastogenic cytokine that induces production by osteoblast
lineage cells of RANKL, the final osteoclastogenic effector
cytokine. IL-17 production is elevated in ovariectomy condi-
tions (Tyagi et al. 2012) and anti-IL-17 antibodies (Tyagi et al.
2014), and IL-17 genetic deletion (DeSelm et al. 2012)
ameliorates bone loss in ovariectomized mice. This process is
further sustained by downregulation of Transforming growth
factor (TGF)b, an estrogen-regulated cytokine, which mediates
7
immunosuppressive effects in part, through induction of regu-
latory T cells (Tregs) that down modulate T cell activation.
This model is presented diagrammatically in Figure 5.
A perplexing aspect of ovariectomy-induced bone loss is the
apparent antigen-dependent nature of T cell activation in this
response. To validate a need for antigens, we performed ovar-
iectomy in mice with silenced antigen presentation due to a
transgenic T cell receptor in all T cells, responsive only to
ovalbumin, a protein not endogenously present in mice (Cenci
et al. 2003). In the absence of antigen presentation, these mice
were fully protected from ovariectomy-induced bone loss.
However, after exogenous administration of antigen (ovalbu-
min), robust bone loss was again elicited by estrogen defi-
ciency. These data confirmed the requirement for antigen
presentation in this response but also suggested that no specific
type of antigen was needed and that any antigen capable of
activating a T cell might suffice. Although a low baseline of
antigen presentation is always occurring even in healthy
humans and animals, recently, the likely nature of the antigens
that are permissive for ovariectomy bone loss have been uncov-
ered. Evidence now suggests that these antigens are gut derived
(J. Y. Li et al. 2016). The gut of humans and animals is a vast
receptor for a myriad of microorganisms collectively referred
to as the gut microbiota, which have the capacity to control
bone mass, as germ-free mice have increased BMD (Sjogren
et al. 2012). The efflux of bacterial antigens into the circulation
is controlled by tight junctions in the cells of the gut wall.
Interestingly, these adhesion molecules are regulated by estro-
gen, and their expression is down modulated by estrogen defi-
ciency (J. Y. Li et al. 2016). The net effect is a leaky gut lining
that allows bacterial and other antigens to enter the circulation.
Attesting to the importance of the gut microbiota in estrogen
deficiency bone loss, germ-free mice do not loss bone mass
after estrogen deprivation but again undergo bone resorption
and loss following recolonization of the gut microbiota (J. Y. Li
et al. 2016). Other studies have shown that treatment with
probiotics can prevent ovariectomy-induced bone loss (Britton
et al. 2014; Ohlsson et al. 2014).
Addition of other factors to this complex network driving
estrogen deficiency bone loss continues relentlessly with identi-
fication of additional participants including insulin-like growth
factor (IGF)-1 (Lindberg et al. 2006) and reactive oxygen spe-
cies to the scheme (Grassi et al. 2007; Almeida et al. 2007).
Although early studies using B cell KO mice did not identify a
role for B cells in ovariectomy-induced bone loss in mice (Y. Li,
Li, et al. 2007), recent studies using sophisticated RANKL condi-
tional KO in the B and T lineage have revealed a partial contribu-
tion of B cells to trabecular (but not cortical) bone loss in
ovariectomy (Onal et al. 2012). As in our previous studies of
murine ovariectomy, this study found no role for RANKL produc-
tion by T cells in ovariectomy-induced bone loss (Onal et al. 2012).
Although studies validating these responses in the human
context remain few and far between, at least one examination
of human bone marrow cells has revealed that both T cells
and B cells produce significantly elevated concentrations
of RANKL, in postmenopausal women compared to
8 Toxicologic Pathology XX(X)

Figure 5. The bone loss of estrogen deficiency. Estrogen (E2) deficiency leads to bone loss though a complex cascade of interacting pathways
involving the immunoskeletal interface. Among the upstream events involved is an upswing in IL-7 production driving CD4þ T cell proliferation
and activation. These events are amplified by production of IFNg that upregulates antigen presentation further stimulating T cell activation. Th1
effector T cells secrete tumor necrosis factor (TNF) which is key to the inflammatory response in estrogen deficiency leading to bone loss. TNF
amplifies receptor activator of NF-kB ligand (RANKL)-induced osteoclastogenesis and stimulates RANKL production through formation of
Th17T cells which secrete IL-17A, a cytokine that promotes RANKL production by osteoblasts. Finally, estrogen deficiency leads to down-
regulation of the anti-inflammatory cytokine TGFb, leading to a decline in formation of regulatory T cells (Treg). Decline in Tregs further leads to
increased immune activation sustaining the inflammatory cascade.

premenopausal controls (Eghbali-Fatourechi et al. 2003).
Treatment of postmenopausal women with estrogen replace-
ment therapy significantly diminished T cell and B cell
RANKL production. Additional evidence in humans has also
been provided by a clinical study examining peripheral blood
T cells which found that RANKL and TNF production was
significantly increased in postmenopausal women with osteo-
porosis but not postmenopausal women without osteoporosis
or in premenopausal women (D’Amelio et al. 2008).
The role of the ISI in postmenopausal bone loss remains
controversial, as not all studies have validated significant
effects of T cells in the response and some mouse and rat
models of T and/or B cell deficiency have produced variable
outcomes (Lee, Kadono, et al. 2006; Lee, Kalinowski, et al.
2006; Sass et al. 1997).
Studies in animal and human systems continue, however,
and new findings are constantly being added to the literature. If
validated, an interesting conclusion may be that estrogen defi-
ciency bone loss has significant hallmarks of an inflammatory
state (Weitzmann and Pacifici 2006).
Bone Formation and the ISI
An interesting finding is that the ISI not only plays key roles in
regulation of bone resorption but also mediates potent effects
on bone formation. In recent year’s potent, immunomodulatory
agents have found therapeutic application in RA (Kuek,
Hazleman, and Ostor 2007), an autoimmune inflammatory
disease characterized by focal destruction of bone and cartilage
in joints and by systemic osteoporosis resulting from release
of inflammatory cytokines into the circulation (Feldmann,
Brennan, and Maini 1996). Much of these cytokines emanate
from activated immune cells, and animal studies have demon-
strated that conditional loss of RANKL production in the
adjuvant-induced osteoporosis animal model of RA protects
mice from bone loss (Kong et al. 1999).
Given the role of inflammation in RA etiology, a T cell costi-
mulation inhibitor cytotoxic T lymphocyte-associated protein-4
(CTLA-4) immunoglobulin (Ig), also referred to as abatacept, is
now Food and Drug Administration (FDA) approved for intract-
able cases of RA. CTLA4-Ig blunts T cell activation and dimin-
ished inflammation and bone loss downstream of the
inflammatory cascades (McCoy and Le Gros 1999).
Because of the role of lymphocytes in the regulation of
basal osteoclastogenesis, an interesting question is whether
long-term therapy with CTLA4-Ig would be detrimental to
the skeleton by causing a RANKL/OPG imbalance and hence
causing collateral damage to the skeleton as was observed in
HIV-induced immunodeficiency. To test this theory, we
injected abatacept into normal C57BL6 mice to examine its
long-term effects (6 month) on basal bone turnover (Roser-
Page et al. 2014). After 6 months of therapy, BMD was
Weitzmann 9

Figure 6. Bone formation by cytotoxic T lymphocyte-associated protein-4 immunoglobulin (CTLA4-Ig) and parathyroid hormone (PTH).
CTLA4-Ig targets and disrupts the CD28 costimulatory interaction with CD80/86. This signal is necessary to activate phosphodiesterase 4b
(PDE4b), which prevents accumulation of Cyclic adenosine monophosphate (cAMP) initiated by T cell receptor (TCR) binding to an antigen
bearing major histocompatibility complex (MHC) I complex on an antigen presenting cell. Sustained cAMP signaling renders the T cell anergic
(dormant) through Protein kinase A (PKA)-induced activation of cAMP responsive element-binding protein (CREB), anergic T cells also secrete
the bone anabolic ligand Wnt10b, following binding of CREB to CREB responsive elements (CRE) in its promoter. Wnt10b binds to its receptors
(LDL receptor related protein (LRP)5/6) on the osteoblasts leading to bone formation. PTH, when administered in an intermittent fashion, is also
able to stimulate Wnt10b production by CD8þ T cells following binding to its receptor (PTH-R), although the intracellular signaling pathways have
yet to be defined. PTH action is also dependent on direct effects mediated on osteoblasts that promote proliferation, differentiation, and increase
longevity of the bone-forming cells.

significantly elevated in CTLA4-Ig-treated mice compared to
mice receiving Ig control. Surprisingly, metabolic turnover
markers did not reveal any changes in baseline bone resorp-
tion; however, markers of bone formation were significantly
increased. These data were confirmed by quantitative bone
histomorphometry, the gold standard for assessment of
dynamic changes in bone formation in vivo (Roser-Page
et al. 2014).
The mechanism of CTLA4-Ig action involves suppression
of CD28 signaling in the T cell. T cell activation occurs
through a process involving 2 separate steps (the dual signal
hypothesis; Sayegh 1999). The first signal involves the presen-
tation of an antigen as part of an major histocompatibility
complex (MHC) complex by an Antigen presenting cell (APC)
to the T cell receptor complex. This is not an activation signal
but in fact renders the T cell anergic (dormant) unless a second
costimulatory signal is received. The second signal is trans-
mitted by the APCs CD80 or CD86 costimulatory ligands to
the CD28 receptor on the T cell. Activation of CD28 alleviates
the anergic block allowing T cell activation, differentiation into
T helper cells, and effector function (Sayegh 1999). CTLA4 is
a physiological molecule produced by activated T cells and
Tregs that acts as a decoy receptor for CD28, blocking CD80
and CD86, and preventing CD28 activation. CTLA4 thus ter-
minates immune responses following resolution of infection, a
necessary requirement to pacify inflammation and prevent a
runaway immune response. CTLA4-Ig is a pharmacological
derivative of the natural CTLA4 molecule and functions simi-
larly to resolve inflammation by rendering T cells anergic
(McCoy and Le Gros 1999).
Interestingly, it has been reported that anergic T cells secrete
high levels of a Wingless integration site (Wnt) pathway agonist
called Wnt10b (Zha et al. 2006). The Wnt pathway is potently
bone anabolic and canonical Wnt signaling modulates many
aspects of osteoblast physiology including proliferation, differ-
entiation, bone matrix formation/mineralization, and apoptosis
(Bodine and Komm 2006). Consequently, a logical hypothesis is
that anergic T cells promote bone anabolic responses by secre-
tion of Wnt10b. To test this model, we quantified Wnt10b pro-
duction by real-time RT-PCR in an in vitro APC assay in which
transgenic T cells (bearing a T cell receptor specific for ovalbu-
min) are cocultured with ovalbumin (antigen)-loaded APC (den-
dritic cells). While T cells alone or APC alone did not express
Wnt10b, T cell engagement of antigen on dendritic cells led to a
small induction of Wnt10b expression. Addition of CTLA4-Ig,
breaking costimulation and rendering the activating T cells aner-
gic, potently upregulated Wnt10b expression by more than an
order of magnitude (Roser-Page et al. 2014).
To further validate this hypothesis in vivo, we treated mice
with CTLA4-Ig and then immuno-magnetically purified T
10 Toxicologic Pathology XX(X)

Figure 7. Parathyroid hormone (PTH) and bone loss. The bone catabolic effects of chronic PTH result from production of tumor necrosis factor
(TNF) by T cells that feeds back though cell autonomous effects to promote Th17 effector cell formation. Th17T cells secrete IL-17A, a cytokine
that binds to receptors (IL-17R) on osteoblasts and osteocytes stimulating receptor activator of NF-kB ligand (RANKL) production that promotes
osteoclastogenesis and bone resorption leading to bone loss. Finally, TNF upregulates CD40 receptor on bone marrow stromal cells. Binding of
CD40 with its ligand CD40L on T cells imparts proliferative and survival cues to osteoblasts.

cells. T cells were cultured overnight, and secretion of Wnt10b
into the medium was quantified by ELISA. Secretion of
Wnt10b protein was significantly elevated by in vivo treatment
of mice with CTLA4-Ig (Roser-Page et al. 2014).
Taken together, the data show a surprising T cell mediated
capacity to not only promote inflammatory bone loss but also a
capacity to potentially repair collateral damage to bone follow-
ing resolution of inflammation through bone anabolic actions
involving T cell secretion of Wnt10b. This model is presented
diagrammatically in Figure 6.
Role of T Cells in Parathyroid Hormone
(PTH)-Induced Bone Formation and
Bone Loss
PTH is a complex and paradoxical factor. As an important
regulator of calcium metabolism in the body, it defends
against hypocalcemia by stimulating the release of calcium
from the skeleton by promoting bone resorption (Weitzmann
and Pacifici 2017). However, primary or secondary hyperpar-
athyroidism causes sustained overproduction of PTH driving
severe osteoclastic bone resorption and skeletal deterioration
(Pacifici 2010). PTH-induced bone loss may be modeled in
mice by the chronic administration of PTH through mini-
osmotic pumps leading to a balance in bone turnover in which
bone resorption outpaces that of bone formation leading to
bone loss. By contrast, when administered in an intermittent
(pulsatile) manner paradoxically, PTH leads to a weak bone
resorptive response that is overshadowed by a robust increase
in bone formation leading to a significant net bone gain. In
fact, teriparatide, a fragment of human PTH injected daily, is
an FDA approved bone anabolic modality for treatment of
osteoporosis through stimulation of bone anabolism in
humans (Pacifici 2013).
Anabolic Actions of PTH and the ISI
Interestingly, anabolic actions of PTH involve the ISI. A tra-
ditional view of anabolic PTH is that PTH induces direct osteo-
blast proliferation and differentiation, activation of quiescent
lining osteoblasts, increasing osteoblast life span by suppres-
sing apoptosis, and down modulation of the Wnt receptor
antagonist sclerostin in osteocytes (Pacifici 2013). Although
the Wnt pathway has been recognized as a key target of PTH
in bone anabolic effects, the source of the Wnt ligands driving
bone formation in this context is poorly characterized and
largely considered to be secreted in a cell autonomous fashion
by osteoblast-lineage cells themselves. However, recent data
suggest that a critical source of Wnt ligand is in fact the T cell.
As with CTLA4-Ig, PTH induces significant production of
Wnt10b from T cells. Attesting to the importance of T cell–
produced Wnt10b, reconstitution of WT T cells into TCRb KO
mice restores the anabolic activity of PTH, while adoptive
transfer of T cells from Wnt10b KO mice fails to rescue the
anabolic activity of PTH (Terauchi et al. 2009). This model is
presented diagrammatically in Figure 6.
Weitzmann
Catabolic Actions of PTH and the ISI
The traditional view of PTH-induced bone resorption is that
PTH targets cells of the osteoblast lineage to increase the pro-
duction of RANKL and decrease the production of OPG. This
rebalances the RANKL/OPG ratio in favor of increased osteo-
clastogenesis and bone resorption (Pacifici 2013).
Evidence for a role of T cells in PTH-induced bone
resorption came from studies almost 2 decades ago report-
ing that transplantation of human parathyroid glands from
patients with hyperparathyroidism into T cell deficient nude
mice failed to induce bone loss, suggesting a role for T cells
in the catabolic activity of PTH (Hory et al. 2000). Follow-
up studies by our group almost a decade later confirmed that
administration of PTH in TCRb KO T cell deficient mice
does not induce a catabolic effect on the skeleton. However,
adoptive transfer of WT T cells into TCRb KO mice res-
cued the catabolic action (Gao et al. 2008). Evidence that
PTH acts directly on T cells came from additional studies in
which the PTH receptor was conditionally deleted on T cells
leading to a failure of PTH to induce bone loss (Bedi et al.
2012). Mechanistically, PTH promotes secretion of TNF
from T cells, which in addition to its amplificatory role in
RANKL signaling and suppression of OPG feeds back on T
cells to simulate their differentiation to Th17 subsets that
secrete IL-17A leading to additional RANKL production by
osteoblast lineage cells. Finally, TNF upregulates CD40, a
costimulatory receptor, on bone marrow stromal cells. Bind-
ing of CD40 with its ligand CD40L on T cells imparts
proliferative and survival cues to osteoblast precursors fur-
ther contributing to the catabolic action of PTH (Gao et al.
2008; Bedi et al. 2010; Tawfeek et al. 2010). This model is
presented diagrammatically in Figure 7.
Conclusions
Our understanding of the ISI and the field of osteoimmunology
continues to evolve rapidly with new discoveries attesting to
the considerable depth of the ISI and the influence of the
immune system on bone. The immune system mediates protec-
tive actions on physiological bone turnover under basal condi-
tions but causes pathological destruction when activated (or
compromised as in immunosuppressive states) and causes
increased fracture incidence in a number of disease states. The
immune response further regulates bone resorption and bone
formation depending on context. The latter discovery may have
important implications for the future development of bone ana-
bolic agents to combat osteoporosis. Teriparatide (anabolic
PTH) is already an FDA approved modality for increasing bone
formation. Our recent work is further focusing on the use of
anergic T cells, as bone anabolic agents and immunosuppres-
sive drugs such as abatacept to further promote bone anabolism
alone or to amplify the effects of teriparatide. With new under-
standing of the integration between immune and skeletal func-
tions, yet additional immune targets are likely to emerge for
amelioration of bone loss in the future.
11
Authors’ Note
The contents of this manuscript do not represent the views of the
Department of Veterans Affairs, the National Institutes of Health, or
the United States Government.
Acknowledgments
The author gratefully acknowledges research support from the
Biomedical Laboratory Research and Development Service of the
VA Office of Research and Development (5I01BX000105) and from
the National Institutes of Health (the National Institute of Arthritis and
Musculoskeletal and Skin Diseases [grant numbers AR056090,
AR059364, AR068157, and AR070091] and the National Institute
on Aging [grant number AG040013]).
Author Contribution
All authors (MW) contributed to conception or design; data acquisi-
tion, analysis, or interpretation; drafting the manuscript; and critically
revising the manuscript. All authors gave final approval and agreed to
be accountable for all aspects of work in ensuring that questions
relating to the accuracy or integrity of any part of the work are appro-
priately investigated and resolved.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: Biomedical
Laboratory Research and Development Service of the VA Office of
Research and Development (5I01BX000105) and from the National
Institutes of Health (the National Institute of Arthritis and Musculos-
keletal and Skin Diseases [grant numbers AR056090, AR059364,
AR068157, and AR070091] and the National Institute on Aging [grant
number AG040013]).
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