Silencing "The Silent Killer": Researchers reveal promising new strategy to prevent and diagnose osteoporosis

Healthy bone structure (left) vs Osteoporotic bone (right). From www.karger.com

Public Interest Note:

             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.

         With such simplicity in describing the biological effects of this disease, it’s surprising that much of the public need celebrities like Sally Field to describe the importance of bone health to the public.  However, with an estimated 54% of postmenopausal women being osteopenic (lower than normal bone mass) and 30% exhibiting full-blown osteoporosis, the bone health industry and advocates for promoting bone health are trying everything they can think of to get people to pay attention this “silent killer”.

            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

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

 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?!

Hsu, YH., et al. “Anti-IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss”. Journal of Experimental Medicine. 208:1849-1861. (2011).

      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.

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!

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!

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:

1. Glick,B. “Historical perspective: The bursa of Fabricius and its influence on B cell development, past and present”. Veterinary Immunology and Immunopathology. 30:3-12. (1991).

2. Yasuda, H. “Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL”. PNAS. 95:3597-3602. (1998).

3. Arron, J. and Choi, Y. “Bone versus immune system”. Nature. 408:535-536. (2000).

4. Yasothan, U. and Kar, S. “Osteoporosis: overview and pipeline”. Nature Reviews Drug Discovery. 7:725-726. (2008).

5. Hughes, DE. “Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow”. J. Clin. Invest. 83:1930-1935. (1989).

6. Opar, A. “Late-stage osteoporosis drugs illustrate challenges in the field”. Nature Reviews Drug Discovery. 8: 757-758. (2009).

7. Sabat, R. “IL-19 and IL-20: two novel cytokines with importance in inflammatory diseases”. Expert Opin Ther Targets.  11:601-612. (2007).

8. Rich, BE. “IL-20: a new target for the treatment of inflammatory skin disease”. Expert Opin Ther Targets.  7:165-74. (2003).

9.Hsu, YH., et al. “Function of interleukin-20 as a proinflammatory molecule in rheumatoid and experimental arthritis”. Arthritis Rheum. 54:2722-2733. (2006).

10. Chen, WY. “IL-20 is expressed in atherosclerosis plaques and promotes atherosclerosis in apolipoprotein E-deficient mice”. Arterioscler Thromb Vasc Biol .26:2090-2095. (2006).

11. Chen, WY and Change, MS. “IL-20 is regulated by hypoxia-inducible factor and up-regulated after experimental ischemic stroke”. Journal of Immunology. 182:5003-5012. (2009).
12: Kong, YY, et al. “OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis”. Nature. 397:315-333. (1999).

How to get the most out of your next vaccine? A full night's sleep may enhance protection against viral infections

from gabby22197.glogster.com

Public Interest Note:

Let’s begin with a simple question: Why do we sleep?  To answer this, we can think of what happens when we don’t get enough or any sleep.  The day after a sleepless night, often we feel irritable, exhausted, unhappy and stressed.  If you are a parent, have a full-time job or a fellow graduate student you are acutely familiar with how precious even a few hours of sleep can be in redeeming your sense of sanity.  The side effects of sleep can become dangerous when the actions of a sleep-deprived individual affect those around him/her.  In March, an air traffic controller at Ronald Reagan Washington Airport fell asleep on the job, leaving two planes alone in the sky trying to land the planes alone.¹ Fortunately, in this case, no one was terrible injured, but the news magnified the public’s awareness and outrage of having to depend on sleep-deprived individuals for our safety.  It’s not surprising the single air traffic controller fell asleep on the job since it was reported this was “his fourth consecutive overnight shift, which runs from 10 p.m. to 6 a.m.” ¹ In fact, the American Academy of Sleep Medicine International Classification of Sleep Disorders classifies this condition as Shift Work Sleep Disorder (SWSD).  SWSD plagues nearly 70% of the 15 million Americans who work shifts between 10 p.m. and 6 a.m.²   It is further estimated that sleep deprived workers cost U.S. businesses $18 billion a year.³  

from cdc.gov

Importantly, sleepiness does not only affect the people who work the “graveyard shift”, but in an increasingly fast-paced society, coupled with a tough economy and technology-rich environment, many Americans are spending less time asleep.  The Center for Disease Control and Prevention (CDC) revealed that more than 35% of the 74,571 adult Americans surveyed get less than 7 hours of sleep a day and 37.9% reported “unintentionally falling asleep during the day at least once in the preceding month.  Alarmingly, nearly 5% of respondents admitted to nodding off while driving at least once in the past month. ⁴   Furthermore, it is estimated that sleep deprivation causes nearly 100,000 vehicle crashes and 1,500 deaths annually in the U.S.⁵ Perhaps, if people more strictly followed the CDC’s recommendation of getting 7-9 hrs of sleep (for adults), such scary statistics will improve and save our economy billions of dollars.  But why does the CDC discuss anything about sleep?  Does sleep have a role in disease prevention?

We know what happens to our bodies when we are sleep deprived: red eyes, achy joints, and sometimes we even catch a cold.  Besides behavioral problems that arise when we are sleep deprived, our immune system becomes compromised as well.  For that reason, the CDC warns, “persons experiencing sleep insufficiency are also more likely to suffer from chronic diseases such as hypertension, diabetes, depression and obesity…” The CDC refers to America’s sleep deprivation problem as an epidemic, a public health concern.  The problem of sleep deprivation in America is so great that since there is even a national organization called the National Sleep Foundation devoted to alerting the public, policy makers and healthcare providers of the consequences related to sleeping insufficiencies. Such agencies are worried that a continuously sleepy nation will result in a sick nation with increased susceptibility to infections and disease-which ultimately will cost the country billions and hamper productivity.  

Sleep Deprivation: The Immunology Behind It

EEG charts and amount of time spent in each sleep cycle, stages 3 and 4 constitue SWS which is also the time in sleep when pro-inflammatory cytokines, IL-1 and TNFalpha peak. Lange, et al. establishes that a full-night's sleep post vaccination BOOSTS your immunological memory, perhaps enhancing the vaccine's effectiveness. Image from seamist.hubpages.com

First, how does one define and measure sleep? There are two types of sleep: non-rapid-eye-movement (NREM) and rapid eye-movement (REM).    There are 5 main stages of sleep beginning with stage 1 in which you are just lying down to sleep, but still pretty conscious.  Stage 2 follows with a lower level of consciousness that develops into stages and 3 and 4.  Stages 3 and 4 are collectively called slow wave sleep (SWS).  Under SWS, the brain is at its lowest level of consciousness and is thought to be involved in restorative functions.  Lastly, REM is the final step in sleep and is when our brains are processing information and dreaming.  When asleep, we typically spend equal parts in REM and SWS.  Sleep stages can be monitored via EEG recordings as each stage has distinctly different frequencies and EEG wave amplitudes.⁶

 In the late 1980’s and early 1990’s, Toth and colleagues performed a series of infections on rabbits and monitored the animal’s sleep patterns during infections.  They tested a virus (influenza), bacteria (Staph. aureus and E. coli ), yeast (Candida albicans) and parasite (Trypansosoma brucei).  Each infection resulted in abhorrent sleep patterns, regardless of microorganism.  In general, the various infections increased the duration of slow wave sleep (SWS). ^6,7  The observation that infection increases SWS, but not other sleep stages, may mean that SWS, in particular, regulates our immune response while we are sleeping.  In humans, one of the most well studied infections to examine in regards to sleep is T. brucei, the parasite that causes Human African trypanosomiasis aka African sleeping sickness.  Infected individuals, usually by the second year of infection, lose the ability to manage their circadian rhythm resulting in the complete loss of sleep regulation and often leads to comatose.  In mice and rabbits, the release of parasites into the bloodstream is associated with acute increases in SWS, however this has not been evaluated in humans. ⁶

So what immune factors are modulated by sleep?  First, it is known that the majority of our immune cells, B and T cells and monocytes reach maximal levels in the blood during the night and are lowest when awake.⁸ In addition, the pro-inflammatory cytokines, TNFalpha and IL-1beta plasma levels also peak while we sleep, being the highest at the onset of SWS.⁸ An increase of either of these cytokines subsequently increases SWS duration, whereas decreases in TNFalpha or IL-1beta blocks SWS.⁶ This appears to be the clearest example of how our sleep patterns may regulate our immune response during infection. Both of these cytokines are critical in the clearance of infection and are some of the most abundant cytokines present during infectious diseases.  Therefore it seems to make sense why an increase in SWS during the diseases mentioned above occurs. 

Interestingly, infection is not the only thing that induces TNFa and IL-1beta release in the brain and plasma.  Sleep deprivation also promotes pro-inflammatory cytokine release, which then instructs your brain to increase the time it spends in SWS.  Alternatively, anti-inflammatory cytokines such as IL-10 is thought to inhibit SWS onset by antagonizing IL-1beta and TNFalpha.  It is also thought the induction of certain hormones such growth hormone (GH) and prolactin promote the release of IL-1beta, since inhibiting these hormones attenuates IL-1beta levels and SWS duration.  Conversely, the anti-inflammatory hormone, cortisol does the opposite, and decreases IL-1beta production.⁶ So it appears that our immune system mirrors our endocrine system in that they both regulate our sleep patterns by releasing pro- and anti- sleep mediators.  

TNFalpha and IL-1beta are generally thought of activators of the innate immune response, such as macrophages and dendritic cells.  Such cytokines act as an adjuvent, boosting the macrophage and dendritic cell's ability to be potent antigen presenting cells (APC).  The main job of an APC, is to display bits of processed pathogen (antigen) to a T cell.  Once a T cell recognizes a particular antigen, it then becomes activated and ready to fight the infection and help B cells secrete antibodies.  Realize that the next time you become ill and start to feel sleepy, your immune system is starting to work and that you should rest so that your body can adequately defend itself against infection.  Ignoring our body's demand for sleep affects the way our bodies fight diseases.  In a few human studies, it was revealed that the number of T helper cells decreased and the level circulating antibodies decreased with sleep deprivation.⁶ Not surprisingly, when the body is pushed to the extreme of total sleep deprivation, the immune system fails completely.  In one study, rats were sleep deprived for 21 days, until near death.  At the end of the 21 days, the rats had suffered from severe weight loss and septicemia with both opportunistic and pathogenic microbes in the blood.⁹ Furthermore, a recent survey of more than a million participants demonstrated a strong correlation between sleep of less than 7 hours a night and increased mortality.¹⁰

  

Sleep Deprivation and Disease: Why This Research Paper Matters

The evidence is clear: our immune system communicates with our brain while we sleep, but researchers have only begun scratching at the mechanisms behind this phenomenon.  This is very exciting and novel research that may provide not only insight into how the body operates, but also instigate new therapeutic approaches in fighting disease.  Although it is well established the role of certain cytokines in maintaining healthy sleep cycles and how the immune system responds to sleep deprivation, little is known about the effect sleep has on specific immune cell function.  The majority of what we know about the interplay between sleep and immune function is correlative.  For example, we know TNFalpha can promote APC function, and we know that TNFalpha levels increase during SWS, but it remains elusive whether sleep-induced TNFalpha promote APC function and thereby program and effective T cell response.  Sleep may represent a crucial part of the immune system that has been largely ignored by the immunological and medical fields.  Perhaps, the notion “Sick and Tired” can be immunologically explained and that greater insight into how sleep affects the body’s ability to clear infection is an underlying mechanism behind the increase of infections among infants and the elderly (acute vs chronic sleep deprivation).  How does sleep impact our ability to properly activate our immune system?  Can sleep play a role in designing better therapies and treatments for people fighting disease?   These questions motivate Jan Born’s research group at the University of Lubeck in Germany to find new ways to make more efficient vaccines…without even changing the vaccine components!  Therefore, as Lange, T. et al illustrates in their recent Journal of Immunology paper, catching a full night of ZZZ's  can make all the difference in getting the most out of your next vaccination.

What the &*%$#! Does the Title Mean?!

Lange, T. et al. “Sleep after vaccination boosts immunological memory”. Journal of Immunology. 187 (2011).

There are two waves of antibody response: a slower, short-lived response during the initial infection followed by a quicker, longer-lived memory response . from click4biology.info

1) The essential point of vaccination is providing people with the antibodies needed to clear infections quickly and effectively.  Vaccines work by administering something that stimulates the adaptive immune response without causing disease.  Vaccines in the U.S. consist of purified proteins (not a whole, intact pathogen) from a pathogen that is injected or inhaled.  When APCs encounter the proteins in the vaccine, they cut the proteins into short peptide sequences.  Once processed, an APC presents the peptide on its surface to a T helper cell, which are named so because they help B cells make antibodies and help innate immune cells to destroy pathogens.  At the moment when the T helper cell recognizes a certain peptide sequence, the T cell begins to proliferate and secrete cytokines that help B cells secrete tons of antibodies- all of which can specifically target the protein that was used in the vaccine.  Vaccines help prevent infectious diseases by assisting our immune system in making the antibodies necessary to clearing an infection quickly.  When we become infected with something that we didn’t vaccinate against, our immune system has to take a lot of time (weeks) to produce the antibodies needed to fight the infection.  Some pathogens are extremely virulent and can cause a lot of harm, even death in the weeks it takes our bodies to properly protect itself against it.  Vaccines therefore help equip our immune system with the weapons (antibodies) it needs, before even heading into battle against a deadly pathogen. 
 2) Of course, this concept of a vaccine works best when the immune system can remember what the pathogen looks like when it encounters it a month, a year, or 10 years from the time of vaccination.  This is immunological memory.  An amazing aspect of your immune system is that when the adaptive immune cells, B and T cells, are activated and proliferate, some of the cells seem to last forever.  These cells are aptly named, memory B and T cells.  These memory cells will last you during your entire life time, always remembering what they are supposed to do when they encounter the specific protein or peptide that stimulated them to become active in the first place.  In order to maximize immunogenicity and achieve immunological memory you need a very good induction of the antigen-presenting cells by using adjuvants and booster shots to bolster the ability of your body to produce a reservoir of effective memory cells ready to kill a particular pathogen over the course of your life!

Compile this information together and we can infer that: Sleep following a vaccination, acts like an adjuvent promoting the development of memory cells that provides long-term immunological protection against a certain pathogen.  

Ready for an adventure? Read on for a guided-tour through the scientific data!

But first: A brief Q and A session regarding the overall experimental approach:

Q) What organism(s) was being tested?

A) All of the experiments presented here were performed on human volunteers.  The sample size consisted of 27 healthy, nonsmoking men with an average age of 26.  The group was on synchronized schedules with the same sleep-wake patterns for the 6 weeks prior to experimental testing.  All sleep-wake activity occurred in a sleep laboratory, and all participants had spent at least one night in the lab prior to experimental testing.  The group was randomly assigned to either the “sleep” or “wakefulness” group. 

Q) How did the “sleep” group differ from the “wakefulness” group?

A) The sleep patterns between the groups only differ the night following their vaccination (which occurred at 8am and occurred 3 times, one in Feb., March, and June. The “sleep” group had lights off at 11pm and lights on at 6:30am in the sleep laboratory.  During this same time, the “wakefulness” group stayed awake in bed watching TV, reading, listening to music or talking to a scientist (fun, no?)  The “wake” group was not allowed to sleep until 8pm the following day.  This may seem a bit extreme and rare that people would be awake for this long following a vaccine shot, but it does adequately distinguish the two experimental groups clearly. 

Q) What vaccine was used?

A) Hepatitis A vaccine (Twinrix, GlaxoSmithKline Biologicals)

Q) What did the researchers monitor throughout the trial period?

A) Four major conditions were tested:

1. Sleep activity via electroencephalography (EEG) to examine sleep stages.

2. Hormone analysis to investigate sleep-related hormone release via i.v. blood collection.

3. Hepatitis A Virus (HAV) - specific T helper cell response

4. HAV - specific antibody response

For immune response, peripheral blood cells (to look at T cells) and serum (where antibodies are found) was collected by drawing blood immediately before vaccination and then 1,2, and 4 weeks after each shot as well as a follow-up at 1 yr. after the first inoculation.

Now let’s dive into the results!

Because the whole basis of this paper rests on sleep activity it is first imperative that the authors of the paper show that the people in the “sleep” group slept normally.  Normal meaning that they spent time in each of the known sleep stages (see the above figure) that fit data that is already well established in the field, with nearly 50% of sleep spent in slow wave sleep (SWS; stages 3+4) and 50% in REM sleep.  There was no significant differences between the three sleep nights post vaccination. 

Over the course of the Hepatitis A vaccination period, blood was obtained to examine the T cell response. This is very important to look at because it is a standard read-out for the quality of a vaccine.  Recall that most vaccines, including the Hepatitis A vaccine, consist of purified proteins that contain immunogenic Hepatitis A viral proteins.  Once these proteins are injected, they enter the blood and tissue where they will be taken up by macrophages and dendritic cells.  Which is very convenient since these cells are antigen-presenting cells (APCs).  So then these APCs digest the viral proteins into smaller peptides and present these peptides on their surface so T cells can “see” the peptide.  If a T cell recognizes that particular peptide, the T cells become divides rapidly producing hundreds of more T cells that all are specific for that peptide.  Scientists can find out how well a vaccine is working by determining how many vaccine-specific T cells there are-the more there are, the more protection will be provided, the better the vaccine.   When a T helper cell encounters a peptide it’s specific for, it not only divides like crazy but it also upregulates an activation marker on its surface called CD40L.   For the purpose of this paper, it’s just important to understand that some proteins like CD40L can be used as a diagnostic tool to track the progression of T cell activation following vaccination.   In this experiment, Lange and colleagues, drew blood from the HAV-vaccinated men in both the “sleep” and “wakefulness” groups, then stimulated the blood cells with a pool of HAV peptides for 6 hours.  In this short amount of time, the only cells expressing CD40L are T helper cells that responded to and are therefore specific for HAV.  In both groups, the % of CD40L+ HAV-specific T helper cells increases.    It is important to note, that quickly following a shot, there is increase followed by a plateau of activated HAV-specific T helper cells and that with each vaccine shot, the T cell response to the vaccine increases; this is the purpose of booster shots.  The surprising result was that by the second HAV shot, there was a significantly lower T helper response in the “wakefulness” group than the “sleep” group.   Intriguingly, this difference lasted even 1 year after the initial vaccine shot with the “sleep” group having 2 times the number of activated, HAV-specific T helper cells than the “wakefulness” group.  Remember that the only major variable between the groups is that the “sleep” group went to sleep the night following the vaccination shots and the “wakefulness” group did not.  This one difference in sleep scheduling contributed to a two-fold difference in T cell activation and response to the HAV vaccine!

 Remember that T helper cells are called “helpers” because they produce cytokines that help other immune cells to become activated and better responsive to the vaccine leading to better protection against pathogens.  To make sure that the HAV-specific T helper cells they were detecting were in fact “helpers”, Lange, et al. measured various cytokines produced from CD40L+ cells in response to the HAV peptide pool.  In all the cytokines tested, the “sleep” group produced significantly more cytokines (Interferon(IFN)gamma, Interleukin(IL)-2, Tumor necrosis factor(TNF)alpha, and IL-4) than the group didn’t go to sleep after getting their vaccine shot.  

 In addition, T helper cells are “helpers” because they “help” B cells to produce and release antibodies (IL-4 helps this, for example).  Because antibodies are one of the major ways vaccines work to protect you against pathogens, the authors of this paper needed to assess whether antibody production is also affected by sleep.  To do this, they need to isolate the serum from the blood cells when they collect the blood samples.  Once they have the serum, which contains proteins and antibodies but not cells, they can look for HAV-specific antibodies-which is basically like searching for a needle in a haystack.  However, researchers can find HAV-specific antibodies in serum by performing an Enzyme-LinkedImmunosorbent Assay (ELISA).  The basis for an ELISA is using a culture plate coated with HAV proteins and then adding serum to the coated plate.  After a brief incubation period, the plate is washed thoroughly, and because antibodies bind extremely well to specific proteins, only the HAV-specific antibodies will stick to the plate, whereas the non-HAV antibodies floating in the serum will be washed away.  Finally, the amount of HAV-specific antibodies present (antibody titer) can be determined easily.  By this method, which is a very common, standard method used by immunologists and medical professionals, they found the people who slept the night following a vaccine shot produced significantly more HAV-specific IgG antibodies compared to those who didn’t get any sleep!

 What is very interesting is that the percentage of HAV-specific T helper cells greatly correlated with the sleep stage #4 compared to the other sleep stages, suggesting that something was present during sleep stage #4 might be regulating this phenomena.  Lange, et al. had found that hormones such as growth hormone (GH) and prolactin levels increased profoundly during SWS period.  Recall, that these hormones induce an inflammatory response, which has been studied extensively as a way the body regulates itself to become sleepy.  What is less known, is if these sleep-associated hormones have a role during the immune response as well.  What these data indicate is that GH and prolactin not only regulates the brain to induce sleep, but they also regulate the immune system to induce a highly activated immune response upon vaccination!  

Furthermore, during SWS, cortisol levels –which is immunosuppressive- is very low.  Given these data, the authors hypothesized that increase in GH and prolactin coupled with the decrease in cortisol levels during SWS provided a boost to the HAV vaccine that the “wakefulness” group did not receive.  In this way, these sleep-associated hormones behave like an adjuvant that further stimulates APCs to activate T helper cells.  By determining an “adjuvant factor” (GH x prolactin divided by cortisol levels), and correlating this “adjuvant factor” with the percent of HAV-specific T helper cells, Lange, et al. discovered the production of GH, prolactin and cortisol can eloquently predict the development of a strong HAV-specific immune response!

  As the authors admit, “the immunoregulatory functions of sleep are not well understood”.  Which makes this particular research so exciting and novel!  Of course, there are lots to expand upon and investigate to better understand how exactly these sleep-associated hormones boost the immune response to the HAV vaccine.  It would also be very interesting to know if sleep affects other vaccines besides HAV and if sleep cycles in women or people who are older or younger than the group tested in this paper provide similar results.  From an immunologist’s perspective, I think it would be valuable to know not only if the development of immune response to the HAV vaccine is significantly better, but also if the immune response is functionally improved because of sleep.  Which will be difficult to do in humans (people are ok with volunteering for a HAV vaccine, but to also volunteer to get infected with HAV? Probably not so great.) But perhaps this aspect could be assessed in mice or by looking at sleep patterns among people who have Hepatitis A and seeing if there is a smaller viral titer in people who sleep more.  It might seem ridiculous, that this paper rests all of its results on the one difference of either sleeping 7.5 hours or not sleeping at all, because it might appear that this data is only applicable if you are an insomniac.  It would be interesting to know what exactly the threshold of sleep (hours) is required to see the phenomena they illustrate in this paper.  However, what this research provides is strong evidence that little to no sleep has a profound effect on the development of your immune response during vaccination.  Interestingly, it would be important to test if sleep-associated hormones have a similar effect during infection with a live virus since this data might provide new insight into why newborns are highly susceptible to such infections (what newborn do you know that sleeps 7+ hours a night?) and why people who work 16+ hour days or graveyard shifts spend more time sick than people who work 9-5.  

Since starting graduate school, I’ve noticed that I have spent more days a year sick than I ever have, I used to think it was because of my increase use of public transportation, living in a bigger city, and interacting with more people (big lab, friends, students of mine, seminars, etc) than I had in my past.  But now, I think that I had been neglecting one of the major differences between life before graduate school and now: longer hours working in the lab generally means less sleep at home.  As if I needed another motivational reason to be done with graduate school!

Lange T, Dimitrov S, Bollinger T, Diekelmann S, & Born J (2011). Sleep after vaccination boosts immunological memory. Journal of immunology (Baltimore, Md. : 1950), 187 (1), 283-90 PMID: 21632713








References and Further Reading: 

1.    Hosford, M. et al. “Air traffic controller asleep on duty at Reagan National, NTSB says”. ABC News. (2011). 
2.    Beers, TM. “Flexible schedules and shift work: replacing the “9-to-5” workday?”. Monthly Labor Review. (2000). 
3.    Awake in Philly Community Education Group. “Fact sheet: shift work sleep disorder”. (2004). 
4.    CDC. “Insufficient sleep is a public health epidemic”. (2011). 
5.    US Department of Transportation, National Highway Traffic Safety Administration, National Center on Sleep Disorders Research, National Heart Lung and Blood Institute. “Drowsy driving and automobile crashes” (2011). 
6.    Bryant, PA., et al. “Sick and tired: does sleep have a vital role in the immune system?” Nature Reviews Immunology. 4:457-467. (2004). 
7.    Toth, LA., et al. “Alteration of sleep in rabbits by Staphylococcus aureus infection”. Infect. Immunity. 58: 1785-1791. (1988). 
8.    Born, J., et al. “Effects of sleep and circadian rhythm on human circulating immune cells”. Journal of Immunology. 158:4454-4464. (1997). 
9.    Everson, CA. “Sustained sleep deprivation impairs host defense”. Am. J. Physiol. 265:R1148-R1154. (1993). 
10. Kripke, DF., et al. “Mortality associated with sleep duration and insomnia”. Arch. Gen. Psychiatry.” 59:131-136. (2002).