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Healthy bone structure (left) vs Osteoporotic bone (right). From www.karger.com |
Put two simple, innocuous words together
and you get a longer word describing a disease that affects 200 million women
worldwide: “Osteo” from the Greek word “osteon” meaning “bone” and “Porosis” meaning
porous. Porous bones. Bone filled with cavities. Bone loss. Weak bones. Fractured bones. These phrases simply describe what
having osteoporosis means.
Hold
up! Did I just go from the Greek word
for “bone” to “killer” in just
over 100 words? Unfortunately,
it’s that easy to connect these two seemingly disparate terms.
Osteoporosis
is often called “the
silent killer” because individuals who have the disease are usually unaware
that they have it until a bone fractures.
Serious bone fractures often leave individuals with osteoporosis
debilitated and can enhance susceptibility to infectious diseases, which can
result in death. In fact, according
the International Osteoporosis Foundation, women over the age of 50 have “a
2.8% risk of death related to hip fracture…equivalent to her risk of death from
breast cancer and 4 times higher than that from endometrial cancer”. Importantly,
the occurrence of bone loss is not limited to having two X chromosomes, as men over the age 50 have a 30% risk of experiencing an osteoporotic fracture,similar to the risk developing prostate cancer.
National
Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) recommends
that people “consider to talk to [their] doctor about osteoporosis” if you have broken a bone and are over the age of 45 (regardless of sex), if you are a 65+ year old woman, are taking certain medications that are known to cause bone loss,have developed poor posture, if you are a woman whose menstrual periods have stopped (or never started upon puberty), if you have anorexia, or have a chronic illness that is known to contribute to bone loss. In most of these cases however,
individuals at risk do not think of consulting their doctor until symptoms
start to appear. Scratch that.
Symptom starts to appear. The major symptom of osteoporosis is
experiencing a bone fracture, which at that point the disease is so progressive
that prevention of the disease no longer applies. Furthermore, treatments may not be able to reverse the level
of bone lost at this late stage in disease development, increasing the chance
of repetitive bone fractures and risk of death in the future.
In
addition to the need for more effective therapies on the market to treat
osteoporosis, there are currently a limiting number of diagnostic tools that
can be easily utilized in the clinic.
Currently, the main tests to assess a person for bone loss is to analyze
his or her bone mineral density (BMD), bone strength, and bone turnover. However, there are limitations to
current diagnostic strategies including correlating data obtained from varying
instruments, bone used for density and strength tests are not always uniform
between testing clinics and techniques used, and in the case of assessing bone
turnover in the blood and urine, these tests do not confidently correlate with
disease progression. Therefore in order to treat this disease
effectively, the need for more precise diagnostics is urgent.
Notably,
the loss of bone strength does not only inflict men and women with hormone
imbalances, but is also caused by various drugs like glucocorticoids and
chronic illnesses such as rheumatoid arthritis, periodontitis and cancer. Additionally, bone loss has been
observed in individuals who live with little to no bone mobilization as seen in
individuals who are confined to a bed and in astronauts who live for long
periods of time in zero-gravity environments.
Bone
loss is becoming of greater importance to confront as a society as large
portion of our population ages into their 50s and beyond. Serious bone fractures can lead to chronic
pain, reduced mobility, disability and an increasing degree of dependence,
which not only negatively impacts our Nation’s workforce, but forcing the
public to pay millions of dollars annually to cover bone-fracture-related
costs. In 2005, it was estimated that over 2 million fractures were treated
costing $17 billion in healthcare costs.
The
International Osteoporosis Foundation predicts the incidence rate to
increase by 50% in 2025, costing upwards of $25 billion to cover medical
treatments in the U.S.
Whether
you care most about the pain people with osteoporosis endure, the deficiency of
effective, accurate diagnostics, or the dismal economics associated with the
disease, the scientists studying the disease need the public’s continuous
support. With such support,
researchers will continue to enhance our understanding of the disease, discover
new drug targets and design novel therapeutics to turn the trend of this
debilitating disease around.
Our Bones and Our Immune System I: How Our Immune
System Relies on Our Bones
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Immune cell development occurs primarily in the bone marrow before cells. From www.ihtc.org |
The
first thing that students in an Immunology 101 course learn is a process called
hematopoiesis. Hematopoiesis is a unnecessarily big
word to describe how a stem cell becomes an immune cell. It makes sense to start a course with
learning this concept, however, it is usually briefly described and skimmed
over by the lecturer. Perhaps it
is because most students are more interested in learning about things that they
have heard of before like: antibody production, organ/tissue donation,
infection, cancer and vaccine development. At any rate, hematopoiesis probably represents
about 1% of the material students will learn about immunology-which is
unfortunate, because there is a lot of amazing things happening when an immune
cell is “born”, that we need more research done to fully understand it all!
So,
if we had to pick somewhere in the body for an immune cell to be born, we might
think of the lymph nodes, spleen or the blood; after all these are the most
popular organs associated with immune function, right? However, surprisingly, the site where
hematopoiesis occurs-where immune cells develop and mature is in the bone. The bone marrow is where all immune
cells, except T cells, spend all their time until they migrate throughout our
blood and tissue ready to protect us from infection and injury. In fact, the “B” in B cell comes from bursa of Fabricius, which was discovered in the late 1950’s
as the organ where antibody-producing cells developed (compared to the
thymic-derived T cells, hence what the “T” in T cell means). If you are wondering why the “B” stands
for some organ you probably have never heard of and not “bone marrow”, it is
because those original 1950’s experiments were performed on birds, and a decade
later it was discovered that mammals don’t have a bursa of Fabricius, but is
analogous to mammalian bone marrow. 1
From
birth to their “adolescence” most of our immune cells are intricately connected
to the bone environment. Since
this revelation, much research has unveiled important factors of hematopoiesis and
the mechanisms that explain how cells emigrate from the bone into tissue. In addition, it is known the bone
environment is a critical component of maintaining a constant supply of immune
cell populations, which is important to clear infections as well as reconstituting
the an immune system after exposure to radiation and chemotherapies. With
over 60 years of research in hematopoiesis, the medical field has greatly
benefited from our understanding of how bone impacts immune cell
development. However, over this
same period, little research has been devoted to understanding the other aspect
of this bone-immune cell relationship: how
immune cells impact to bone development and health.
Our Bones and Our Immune System II: How Our Bones
Rely on Our Immune System
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Cytokines associated with the chronic inflammatory disease, rheumatoid arthritis are used to model the complex nature of the immune system regulating bone development. From R&D.com |
The
idea that immune cells impact bone development seemed to happen serendipitously
in the 1990’s, beginning with the discovery that one of the major cell types
that make up our bones was derived from a hemopoetic cell. Soon, Udagawa and colleagues published their
research findings that monoytes can turn into bone cells called osteoclasts.
Monocytes
are unique immune cells because unlike B cells, for example, which will always
be B cells, monocytes can further differentiate into a variety of immune
cells. Monocytes are able to do
this because they are acutely sensitive to changes in their microenvironment
and highly responsive to a range of stimuli that instructs the monocyte to turn
into a different kind of cell.
Osteoclasts, macrophages, dendritic cells, and microglial cells all
derive from a monocyte precursor.
What makes each of these cells unique is what stimuli a monocyte
senses. For example, in vitro, to
generate macrophages (which I do on a weekly basis), all you need to do is
isolate bone-marrow cells or monocytes and throw in some macrophage-colony
stimulator factor (M-CSF), wait a week and you’ve got macrophages! If you want dendritic cells, do the
same thing, but this time in addition to M-CSF add some IL-4. Back when it was just learned that
osteoclasts come from monocytes, a lot of research was done to figure out two
major things: 1) what exactly an osteoclast was and 2) what a monocyte needed
to become a bone cell.
An
osteoclast is a macrophage-like cell.
This seems to be post heavy with Greek terminology, so I’ll keep it up
by describing a macrophage as a cell that is very big (aka “macro”). An osteoclast is very similar in that
they too are huge, in fact they are often referred to as (and I am not making
this up): “giant cells”. They are
so big because are created when developing monocytes fuse into a single, giant
cell. One of the most
distinguishing traits of an osteoclast is that they are multi-nucleated. Osteoclasts differ from macrophages in
many ways; however, including first and foremost-they stick specifically to
bone instead of residing in tissue.
Moreover, unlike other monocyte-derived cells, osteoclasts are capable
of resorption, the process of
degrading bone by pumping large amounts of hydrogen ions into the bone. Accumulation of these ions and other
enzymes from the osteoclast into the bone causes the bone matrix to acidify and
break down, which results in cavities in the bone. Cavities typically have a negative feeling associated with
them, but destruction of bone is not always a bad thing. Think of what would happen if
your bones just kept growing. You
might develop strange bone abnormalities or have problems acquiring enough
nutrients to keep the excess of bone healthy. So bone development, like everything in biology is a tightly
regulated process. In order to understand
how errs in bone development lead to osteoporosis, it is important to
appreciate how osteoclasts are generated, as they are the key to the pathology
associated with bone disease.
Firstly,
what a monocyte needed to turn into an osteoclast proved to be more complicated
than anticipated. Initially it was
difficult because there were two groups of researchers working separately on
something that unbeknownst to them at the time, was in fact the same
thing. There were the people
studying bone trying to figure out more about how bone developed and there were
the people working on the immune system trying to figure out how monocytes
turned into osteoclasts. For
example, in the 1990’s, two important discoveries were made: First, the bone
physiologists identified a protein, expressed by osteoblasts and stromal cells
that was proven to be essential to osteoclast development- they called this
protein osteoclast differentiation factor (ODF). Secondly, the immunologists were please to announce the
discovery of receptor activator of nuclear factor kappa-B ligand (RANKL), which is produced by T cells
and was determined to be the second stimulus, in addition to M-CSF a monocyte
needs to turn into an osteoclast.
It was later revealed that both groups of researches had actually
discovered the same protein such that ODF is identical to RANKL! 2 Realizing this, Drs. Joseph
R. Arron and Yongwon Cho quickly coined the term ‘osteoimmunology’ to be “used to describe the interface between
these two disciplines”. The authors
of the article go on to explain, “Without a better understanding of this
interface, it will be difficult to prevent or treat many common diseases that
affect both bones and the immune system”. 3
Treatments for Osteoporosis: Why this research
matters:
Bones
are “Often thought of as a rigid, unchanging entity, skeletal bone is actually
the result of a dynamic process” involving a balancing act between the activity
of bone formation by osteoblasts and
bone destruction by osteoclasts.
The harmony between these two processes is essential for maintaining
strong bones. It is when the rate of bone resorption exceeds the rate of bone
formation that leads to osteoporosis. Why does this happen? How can we slow the rate of
resorption? What factors are present that promote osteoclast function to
degrade bone? If we knew the answers to these questions we could better treat
bone-loss related diseases or prevent the incidence of bone fractures!
The
“gold standard” for osteoporosis therapy is the use of bisphosphonates, which
first entered the drug market in the late 1990’s by Merck [Fosamax aka
alendronic acid] and Sonofi-Aventis/Proctor and Gamble [Actonel aka risedronic
acid]. Currently drugs that are
classified as bisphosphonates to treat bone loss represent more than 70% of the
market with the expansion of generics and drugs that have improved dosage like
Boniva [zoledronic acid, Novartis]. Another big reason for dominance of these drugs in the market is the history of
50 years of research detailing how how bisphosphonates regulate bone
development. 4
So
what are bisphosphonates and how do they work? They are simple, small chemicals that consist of two
phosphonate groups (PO3-) linked together by a carbon atom. These chemicals are particularly good
at binding to calcium, so when they enter the body, bisphosphonates concentrate
in the bone. Drugs in the
bisphosphonate class decrease bone resporption because once they enter a cell,
they inhibit the cell’s ability to metabolize energy (ATP), which leads to a process
called apoptosis or cell death.
Because osteoclasts are intimately bound to bone and have some
macrophage-like properties like gobbling things up, bisphosphonates are
particularly good at killing osteoclasts, thereby reducing bone resorption.
5
However,
to no surprise, there are multiple risks and side effects associated with this
class of drugs including: gastrointestinal irritation, oeophageal irritation,
hypocalcaemia, renal irritation or more rarely: osteonecrosis of the jaw and
atrial fibrillation. There many
reasons why there is a need for newer drugs on the osteoporosis market. For one thing, bisphosphonates and
other available drugs are not very specific to osteoclasts. Any cell has the potential to take up a
small chemical and be affected by it.
In addition, although the majority of the drug will work at its target
site (bone), some amount of the drug is likely to influence the function of
off-target sites as well.
Furthermore, these small chemicals are given orally introducing a high rate
of non-compliance by patients, decreasing the potential efficacy of the drug.
4
Second
to bisphophonates in populatrity is a class of drugs called selective estrogen
receptor modulators (SERMs). 4 Although a decrease in estrogen tends to correlate with
the onset of osteoporosis, which one reason why the prevalence of this disease
is greater in post-menapausal women, there are a variety of reasons why SERMs
are not the most effective treatment for bone loss. For one, estrogen is not the only factor influencing the
development of osteoporosis, and does not correlate in every case. It is difficult to determine what, if
any, is the threshold for estrogen that a women should have to prevent
bone-loss. Furthermore, SERMs are
not as effective for women with sufficient estrogen levels or men that suffer
from the disease.
Over
the last few decades there has been strong developments made to generate drugs
that have increased specificity, direction towards the target site, and
effectiveness. Furthermore, there
is a great desire to not only treat bone loss, but to develop better diagnostic
tools to diagnose bone loss, before the disease triggers a debilitating
fracture. According to a recent review regarding the osteoporosis drug
pipeline, “the osteoporosis market has not been a major target for
innovation. R&D activities are
targeted at improving existing dosing regimens with the goal of reducing the
pill burden in a highly medicated population”. At the top of the list for
innovative, promising treatments for osteoporosis is an immunotherapy drug
called Denosumab
(Brand name(s): Prolia®, Xgeva®). Denosumab is an antibody that binds
tightly to RANKL. Denosumab is
classified a blocking or
neutralizing antibody because when it binds to its target, it attaches to
the target very specifically and very tightly, blocking the function of the
target. Denosumab, binds tightly
to RANKL, preventing RANK from activating it. Because of this, the side effects can be potentially
very limited, compared to chemical inhibitory drugs. 6 Furthermore, because it is injected
subcutaneously 1-2/year it enhances patient compliance since patients would not
be required to remember to take oral pills, like for bisphosphonates. Moreover, in mice it has been shown to
inhibit osteoclast development and bone resorption. Just last
month, Denosumab received U.S. FDA approval for human use to treat bone loss in
patients with breast and nonmetastatic prostate cancer. It is currently in clinical trials
expand its use to other osteoporotic diseases. Amgen is developing Denosumab.
In
order to enhance the number of promising therapies in the R&D pipeline,
more research focusing on the basic immunological understanding of the disease is
needed. By fully understanding the biological mechanisms behind disease
development, we can then develop innovative, effective therapies and diagnostic
tools to help answer these questions:
·
Is there a way
to not only delay the onset of disease, but also prevent it?
·
Is there a way
to not only reduce the chance of bone fracture, but to prevent it?
·
Is there a way
to improve our method of detecting disease, as individuals with slightly low
bone mass may go under-the-radar of current bone density measurements?
Without promoting our understanding of how disease develops, we are stuck, unable to move forward in a world where we have safe, available therapies to treat, cure and prevent diseases like osteoporosis
What the &*%$#! Does the Title Mean?!
1. IL-20 is a
cytokine, a secreted molecule
that regulates inflammation. What
exactly it does is a topic of current research, since it was only discovered 10
years ago. We know that monocytes
are potent secretors of IL-20 and that the cells that express the receptor for IL-20
include: keritonocytes (skin cells) and endothelial cells (line blood
vessels). Much of the work done to
understand IL-20 biological function has been focused on what IL-20 does to
cells expressing the IL-20 receptor.
For this reason, most of what we know about IL-20 is from a variety of
skin and blood vessel-related diseases.
For example, one of the earliest studies revealed that keritonocytes
rapidly divide and produce lots of potent pro-inflammatory cytokines including:
monocyte chemotactic protein-1 (MCP-1) and TNF-alpha.7 In addition,
generating mice that overexpress IL-20 results in skin abnormalities such as
thickened epidermis and a wrinkled appearance. These novel discoveries initiated scores of experiments
evaluating the role of IL-20 in inflammatory diseases of the skin like
psoriasis and are currently a top therapeutic target to suppress cutaneous
inflammation. 8 In addition to psoriasis, a similar inflammatory,
disease-promoting role of IL-20 has been established in rheumatoid arthritis9,
athersclerosis10, and stroke 11.
2. Monoclonal
antibodies are
antibodies that are not only specific for the same antigen, but also recognize
the exact same amino acid sequence on that antigen. Normally when you mount an immune response against something
like a bacterium, for example, your antigen presentation cells (APCs) will ingest the bacteria and chew up into tiny bits. If the APCs stopped there, your B cells wouldn’t get
effectively activated and you wouldn’t be able to make lots of antibodies to
attack the bacteria and protect you from future infections. In order to get these amazing antibody
benefits, the APC must also present those chewed-up bits of bacteria on their
cell surface so that your adaptive immune system (T and B cells) can wake-up
and realize that there is an infection going on. You have millions of T cells in your body, all expressing a
unique T cell receptor to recognize a single stretch of amino acids of an
antigen-those bits put on display by APCs. Once a T cell recognizes its specific antigenic sequence, it
becomes activated and ready to help B cells make loads of antibodies. When
a B cell receives T cell help, it rapidly proliferates generating hundreds of B
cells all instructed by the T cells to make an antibody against that specific part
of the bacterium that the T cell just saw. With a little nudge from a T cell, a single B cell divides
into thousands of B cells. When a
B cell divides, it is also going through a dramatic, incredibly unique process
called affinity maturation. With each division, a B cell is
rapidly mutating the DNA that codes for the antibody’s specificity. This process results in the production
of thousands of B cells that are all able to recognize that specific bacterium,
but with slightly different affinities.
But remember that T cells are not all the same and have a range of
specificities to any particular antigen, so a variety of different T cells are
doing this to different B cells, which quickly multiplies the number of
different kinds of antibodies generated during an infection. It’s like inviting a couple friends
over for a drink, but then each of your friends decide to invite some of their
friends and their friends invite some of their friends and so on. Before you know it you have a house
full of different people partying it up, having a great time. This
is essentially the same thing that happens in your body during an infection, so
the next time you’re sick, remember that you have your partying B cells to
thank for your swollen lymph nodes (and your ability to beat the infection)!
But what makes our immune system so amazingly
effective is that these B cells must compete for available antigen to promote
their survival and ability to secrete their antibodies they’ve just generated
through affinity maturation.
This selection process weeds out the B cells that made ineffective,
poorly binding antibodies, so that your body is left with the a handful of the
most selective antibodies that bind the tightest to the pathogen. This group of top-tier antibodies is
the end result of a normal immune response and is collectively called, polyclonal antibodies, since
there is still some variation among the specificity of antibodies generated,
but they all derived from the same initial B cell.
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Monoclonal antibody generation in the lab. |
So how do
you get from a variety of antibodies with varying specificity (polyclonal) to a
variety of antibodies with the exact same specificity (monoclonal)? You do it in the lab. Monoclonal antibody production is not something your body
does naturally, because when your body is at war with a pathogen, it’s best to
have as many weapons as possible to use in the attack, right? But, in research, scientists want to
limit variables and find the most specific weapon to develop therapy. In order to do this, all the antibodies
have to be the same. Exact same B
cell. Exact same affinity. Exact same specificity. Exact same antibody made. You get the idea. Ok, so in order to do this, you first
find something you’re interested in making antibodies against, say that
bacterium that infected us in the previous example. In order to make antibodies against the bacterium, you need
an infection, it doesn’t need to be robust or cause disease, but enough to
stimulate B cells, like in a vaccination.
In the
lab, this is usually done in mice.
After a few weeks since the immunization with the bacterium, the mice
will have hundreds of B cells activated with a polyclonal antibody
repertoire. The spleen is
isolated since that’s where the majority of B cells reside. The antibody-producing B cells are then
isolated and cultured with an immortal cell line to create hybridomas. B
Cell Hybridomas are generated so that these B cells can live indefinitely
so that monoclonal antibodies can be obtained, since natural B cells don’t live
for very long in culture. After
selecting for the B cells that have successfully fused with the immortal cell,
then scientists split the hybridomas into a culture plate so that there is only
1 B cell hybridoma per well. Then,
they stimulate each hybridoma with that same antigen you used to immunize the
mice before. Because there are no
variability from T cells and the culture is in single-cell suspension, the single
cell in culture dish will clonally expand, producing identical antibodies. Specific antibodies based on affinity
strength and specificity is further selected for using biochemical techniques,
to result in the acquisition of purified monoclonal antibodies. These monoclonal antibodies then can be
used as reagents for experiments (FACS, Immunoprecipitation,
Immunohistochemistry), blocking antibodies for therapies and experimental
use, or immunotherapies for individuals who cannot mount their own immune
response (i.e. Synagis to
treat infant respiratory syncytial virus)
Now, with the information above, we can infer:
Antibodies were generated to produce something
that is highly specific for the pro-inflammatory cytokine, IL-20. This paper is going to provide data revealing that when this anti-IL-20 antibody binds to
IL-20 it blocks monocytes from developing into osteoclasts, thus preventing
osteoporosis.
Ready for an adventure? Read on for a guided-tour
through the scientific data!
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Using fluorescent microscopy and nuclear stains, such as DAPI (blue) scientists can visualize multinucleated osteoclasts, how cool is this picture? From microscope.olympus-global.com |
Although, IL-20 signaling
has been associated with a variety of diseases that share bone-loss as a
symptom, there was yet any data regarding the levels of IL-20 produced in
osteoporotic patients. Therefore,
one of the first things that Hsu and colleagues wanted to assess was whether
IL-20 cytokine levels were elevated in individuals suffering from
bone-loss. To do this, they
compared IL-20 concentration in patient serum between 33 41-60 year-old healthy,
62 41-67 year-old osteopenic and 37 40-81 year-old osteoporotic women. Indeed,
circulating IL-20 was significantly increased in women who were clinically
diagnosed with either osteopenia or osteoporosis. Of note, this assessment
was limited to Asian women (as the research group is based in Taiwan) and women
with known metabolic bone diseases, diabetes, cancer, renal disease,
athrosclerosis, using steroids or medications known to influence bone-loss were
excluded in this study.
To
examine the role of IL-20 in bone-loss pathology, the researchers utilized a
common mouse model that mimics the disease as seen in humans. The osteoporosis mouse model is
achieved via ovariectomy (OVX).
Similar to what was observed in osteoprotic women, IL-20 found in the
serum of OVX mice was significantly greater compared to normal female mice,
thereby suggesting that the OVX mouse model does, in fact, adequately represent
osteoporosis disease symptoms (rise in IL-20) similar to the women clinically
diagnosed with osteoporosis.
In
a recent study, Hsu and colleagues studied the role of IL-20 in rheumatoid
arthritis (RA). For those
experiments, they generated mouse anti-human IL-20 monoclonal antibodies and
found that these antibodies inhibited IL-20 signaling in RA, which lessened the
severity of the disease. Now, Hsu,
et al. wanted to know if this IL-20-specific antibody would have similar
ability to reduce the severity of disease symptoms in osteoporosis. They
call this anti-IL-20 monoclonal antibody 7E. In the OVX mouse model, they found that when they treated
these mice for 2 months with 7E, the levels of IL-20 dropped to that of non-OVX
females or OVX mice treated with estradiol, which serve as positive controls
of healthy mice.
Furthermore, analysis of bone morphology and bone mass density (BMD)
revealed that OVX mice treated with the anti-IL-20 antibody, 7E, exhibited less
bone loss than mice that did not receive 7E. It is important to note, this
research team was quite thorough in their experiments by including a number of
positive and negative controls to justify that the observed effects with 7E was
specific to IL-20.
To
show that these effects were specific to 7E and not to any injected antibody,
the scientists also included a control group of OVX mice treated with
non-specific antibody (mIgG), which did not attenuate IL-20 levels or
bone-loss. Moreover, by using ELISA in which they coat a
culture plate with the 7E antibody to “capture” serum cytokines followed by a
secondary antibody to detect a variety of cytokines with similar structure to
IL-20, Hsu and colleagues demonstrated that the 7E monoclonal antibody they
generated was specifically recognizing IL-20.
At
this point, there is data to suggest that IL-20 has a role in promoting
bone-loss, since levels of this cytokine increase with disease and neutralizing
the cytokine with an IL-20-specific antibody ameliorates disease symptoms. However, HOW IL-20 is doing this and what cells are responsible for the
IL-20-mediated effects are completely unknown.
Given
what is currently understood about the cells involved in bone resorption, the
research team started to focus on osteoclasts. First, Hsu, et al. tested whether 7E affected the ability to
generate osteoclasts from hematopoietic stem cells (HSCs). As described above, these precursor
cells require M-CSF and RANKL in order to differentiate into osteoclasts. Regardless of when 7E was given to HSCs,
prior to or at the same time as M-CSF/RANKL, 7E inhibited osteoclast formation
(look for the giant cells!):
In
addition to the defect in giant cell formation, treatment with 7E reduced the
expression of a variety of osteoclast markers including: RANK, c-Fos, Cathepsin
K, NFATc1 and TRAP. These data show 7E can be used to block
osteoclast formation in vitro, which for the antibody to have an effect, it
needs to bind IL-20 present in the culture. Because there were no other cells present in this culture,
these data indicate that osteoclasts are capable of producing and sensing IL-20
themselves!
To
determine if these precursor cells were capable of producing IL-20, Hsu and
colleagues looked at IL-20 and IL-20 receptor expression in HSCs. Only in the presence of M-CSF, did HSCs
express IL-20 illustrating that in addition to giant-cell formation, M-CSF
treatment induces IL-20 production in these cells. Furthermore, when these M-CSF-derived osteoclast precursor
cells (monocytes) were treated with exogenous IL-20, RANK expression
significantly increased, suggesting that cells developing into osteoclasts
produce IL-20, and respond to IL-20 by upregulating RANK on their cell
surface. Recall that RANK, is the
receptor for RANKL and is required for osteoclast development and
function. The IL-20 neutralizing
antibody, 7E, was able to greatly diminish RANK expression by osteoclast
precursors, despite the presence of IL-20 in the culture. These
data provide compelling evidence that IL-20 promotes osteoclast development and that IL-20 may promote osteoporosis.
These
cells may enhance RANK expression on their cell surface to enhance their
ability to sense and bind available RANK ligand (RANKL) in the bone
environment. The idea is that the
more RANK or RANKL available, the more monocytes are developing into
osteoclasts that lead to degradation of bone. To further assess how IL-20
affects in bone development, the research team looked into how IL-20 affects
osteoblast function. In an
osteoblastic cell line culture, IL-20 enhanced osteoblast signaling activity
and RANKL expression. Currently, the understanding of the induction pathway of
osteoclast development begins with RANKL stimulating RANK on M-CSF-stimulating
osteoclast precursor cells. But here, Hsu, et al. provide data
showing that something upstream of RANK/RANKL is controlling RANK/RANKL
function and therefore ultimately controlling osteoclast development--IL-20! These are the first data indicating how
RANK and RANKL expression is modulated by a soluble factor associated with bone
disease!
To
further investigate the role of IL-20 signaling in promoting osteoporosis, the
research team generated IL-20R1 knockout mice. By removing the ability of IL-20 to stimulate signaling
cascades responsible for osteoclast development, they observed that aging mice
lacking IL-20R1 had increased BMD compared to wild-type counterparts. In addition, mice lacking IL-20R1 had a
defect in osteoclast development from monocytes and were unresponsive to IL-20
or 7E treatment, thus further accentuating the specificity of 7E for
IL-20. So perhaps, humans, like
mice may lose the ability to maintain high levels of IL-20R1 thereby increasing
the chance of developing more bone resorbing osteoclasts. However, Hsu and colleagues did not
investigate whether similar data regarding IL-20 receptor expression is
observed in humans.
Lastly,
Hsu, et al. went back to see if the IL-20R deficiency provided similar results
as their IL-20 blocking antibody, 7E in the development of osteoporosis. To do this, they performed
ovariectomies (OVX) on IL-20R1 deficient (IL-20R1-/-) and IL-20R1 sufficient
(IL-20R1+/+;+/-) mice. Similar to what they observed in mice
treated with 7E, OVX mice lacking IL-20R were protected from bone-loss!
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This diagram depicts what was known about osteoclast function, what Hsu, et al. were able to add was data to answer the question: WHAT regulated RANK/RANKL expression? Hsu, et al. showed for the first time that IL-20 upregulated RANK/RANKL and that an IL-20-specific antibody could block this expression and IMPROVE osteoporosis symptoms! Importantly, they also demonstrated that IL-20 may be used as a potent diagnostic marker to indicate the presence of osteoporosis in humans!! Figure from HealthPlexus.net |
By
utilizing a variety of methods including generating IL-20-specific blocking
monoclonal antibodies and generating IL-20R-deficient mice, Hsu and colleagues
convincingly provide data that are the
first to describe the role and function of IL-20 in promoting
osteoporosis. Importantly, this is
also the first report describing the positive correlation between IL-20 serum
levels and bone-loss disease. These findings lend insight and development of better
diagnostic tools that may detect the onset of bone-loss earlier than current
diagnostic methods. More research
is needed to elucidate the kinetics of IL-20 production in order for IL-20 to
be effectively used as a biomarker
to track the onset and progression of bone disease.
The
current clinical treatments for bone-loss include drugs that largely target
estrogen, calcium, or RANK/RANKL signaling. The disadvantages to the former two are highlighted in the
above section; however it is important to note that in mice, genetic deletion
of either RANK or RANKL results in severe osteopetrosis (increased bone mass)
and the utter loss of osteoclasts.
Which although may cure osteoporosis, it leads to poor bone homeostasis
and may cause detriment to immune cell and bone development. Because of these findings, there is
potentially a danger in developing osteopetrosis with individuals with
RANK/RANKL mutations or using drugs that completely block RANK/RANKL function.12
Because of this, it is imperative for
researchers to continuously unveil new understandings in signaling pathways and
be scavenging for new therapeutic targets in order to achieve the most
effective drugs with the least amount of harmful side effects. Studies, such as this one lead by
Ming-Shi Chang at the National Cheng Kung University in Taiwain, is a testament
to the encouraging role academic scientists play in developing promising, novel
immunotherapies to help treat millions of people around the world.
Hsu YH, Chen WY, Chan CH, Wu CH, Sun ZJ, & Chang MS (2011). Anti-IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss. The Journal of experimental medicine, 208 (9), 1849-61 PMID: 21844205
References and Further Reading: