Hey Virus: Replicate Here!

The innate immune system, that part of the immune system that responds to non-specific signs of pathogen infection, faces a quandary when mounting a response against a pathogen. On one hand, it must limit the replication of the pathogen to prevent spread throughout the body. On the other, it must facilitate the development of a specific adaptive immune response, which can clear the pathogen from the body and lead to immunologic memory. It might seem like both of these are reasonable tasks, but the quandary lies in the fact that the adaptive immune system must be activated by a certain amount of antigen (proteins derived from the pathogen that can be effectively targeted by the immune system) in order to elicit an effective response. Low levels of antigen lead to weak responses, whereas high amounts of antigen induce strong responses. Thus, if the innate immune system does the first part of its job too well and largely prevents virus replication, there will not be enough viral antigen present to stimulate a robust adaptive response. So how does the immune system get around this predicament? A new paper in Nature Immunology by Nadine Honke and her colleagues sheds some light on this question (1).

            The major weapons in the innate immune system’s arsenal are small, secreted proteins called interferons. When cells in the body detect a pathogen, they produce interferons, which alert the surrounding cells to the impending danger. These “warning flags” bind to interferon receptors found on the surface of all cells, and induce the expression of a large number of genes, termed interferon stimulated genes (ISGs), which can combat the pathogen through various means. Interferons also initiate mechanisms that facilitate the adaptive immune response, by increasing the presentation of specific antigens to the T cells and B cells that comprise the adaptive immune system. To effectively stimulate T cells, however, requires a sufficient dose of antigen: prior studies have indicated that over 20,000 copies of the antigen are required for an antigen-presenting cell to induce a robust T cell response (2). So, how can the innate immune system effectively prevent the spread of a pathogen while still providing enough “grist” to be presented to naïve T cells? Honke et al began to answer this by examining a particular subset of innate immune cells, metallophilic macrophages.

Sleep, Our Immune System, and Napping

Attention college students. Which of the following scenarios sounds familiar to you?

1) You have just pulled 3 all-nighters and did terribly on your exam because you could not stay awake.

2) You have just pulled 3 all-nighters, you made the mistake of taking a “nap”…and now all you can do for the next few days is sleep. So yeah, you got that extension on the paper, but are still unable to turn it in on time.

Yep, we’ve all been there. But rejoice, we finally have an alibi. It’s really not us—it’s our immune systems! …Or is it? Faraut, et al., in this paper, have hown a link between sleep deprivation and our immune system. Essentially, they have confirmed that when we are sleep deprived, certain aspects of our immune systems flare up. That flaring up can only diminished with napping or with an extended period of “recovery sleep”. In fact, the authors show that napping is even more effective at regulating immune responses than extended sleep.

For this study, Faraut, et al. plucked out 40 young, healthy men and subjected them to a series of screening tests (after paying them, of course). First, all 40 participants had to fulfil the following criteria:

They had to be:

1) Non-smokers

2) Not regular nappers (but slept regular nights for 7-9 h)

3) Between 19-25 in their BMI

4) 18-27 years old

5) Free of diseases and sleep-complaints

They were also put through various tests: psychiatric, as well as medical, screenings, sleep questionnaires, polysomnography to monitor their sleep patterns, and CRP and leukocyte concentrations to determine each individual’s healthy, non-inflammatory levels. The actual experiment itself was carried out as follows (Fig. 1):

[ Fig. 1 ]

After the third “Recovery” day, Faraut, et al. looked at 4 things: the peripheral blood leukocyte count, the level of peripheral blood inflammatory and athergenesis biomarkers, the individual sleepiness of the participants and their cortisol levels. The authors saw that in all experimental groups, the leukocyte (white blood cell) and neutrophil (inflammatory white blood cells) levels increased significantly after the “restricted” night in which they had only 2 hours of sleep. However, the group that had a half-hour nap along with their recovery sleep (Group 2) and the group that had extended sleep (Group 3) were able to decrease this spike in leukocyte/neutrophil expression. This downregulation of leukocyte levels was not evident in the group that had the recovery sleep alone (Group 1).

The researchers performed a sleepiness assessment test based on the Stanford Sleepiness Score, and the Maintenance of Wakefulness Test. The results of this test mirrored the leukocyte/neutrophil results. They saw that across all three groups, there was a uniform increase in sleepiness at 1:30 pm after sleep restriction. In looking at each recovery group, they noted that Group 1 was a lot sleepier at 3 om than Groups 2 and 3. However, Group 3, which had the extended sleep period, was sleepier compared to the nap-takers (Group 2). They also saw that after restriction, the nap-takers had fewer onsets of sleep (none), whereas extended sleepers and normal sleepers (Group 1) had 1 and 2 onsets of sleep, respectively. This indicates that napping is more condusice to restoring alertness after a period of sleep deprivation, which is consistent with other studies that have been done on napping (1).

The authors also monitored what they call the subject’s “sleep architecture”, which is the kind of sleep an individual experiences. There are basically two kinds of sleep: slow-wave sleep (SWS) and Rapid-Eye Movement (REM). SWS represents a period of deep, restful sleep, whereas REM indicates a lighter sleep experience (dreams, which are often rare during deep sleep, occur during REM). Since results indicate that napping seems to provide a better path to recovery than just 8-hours of recovery sleep, or even an extended, 10-hour recovery sleep, Faraut, et al. determined that simple a half-hour of deep, SWS sleep can drastically reduce the homeostatic pressures of sleep. Therefore, deeper sleep, especially in short bouts, prevents our immune system from going into overdrive and our bodies from trying to regulate that abrupt change.

So how does our immune system play into all of this? Well, sleep deprivation triggers various inflammatory responses from our immune system. Studies have shown that pro-inflammatory cytokines such as IL-8, IL-6, and TNF-a are secreted following chronic partial or total sleep deprivation (2, 3). Additionally, these inflammatory responses can bring about cardiovascular disease (2, 4). Faraut, et al. were not able to see significant increases in IL-8, CRP, or fibrinogen as they had expected. They did manage to see, on the other hand, increases in MPO (myeloperoxidase) levels immediately after sleep deprivation. This is significant because MPO is an peroxidase enzyme that defines the granulocyte function of neutrophils (ie., neutrophils release it to chew things up). MPO also catalyzes the formation of oxidizing agents that eventually bring about atheromatous plaque.

Sleep deprivation and the subsequent increase in peripheral blood leukocytes, therefore, has implications in causing cardiovascular mortality. This is consistent with studies that have shown that there is a higher risk of cardiovascular diseases in shift workers (5). Conversely, midday napping has also been shown to be inversely associated with coronary mortality (6).

Interestingly enough, a study by van Mark, et al. (7) speculates that chrinic sleep dept (almost like insomnia) is common in shift workers, possibly as a protection mechanism against an overstimulation of pro-inflammatory immune mechanisms. This makes sense because we are able to 3 to 4 consecutive all-nighters, and feel relatively fine while doing so. This is due to the suppression of the homeostatic actions of our bodies. However, as soon as we go to sleep, we seen to never be able to wake up. This is likely due to the fact that our immune responses are finally kicking in, and our bodies are naturally trying to make up for the cytotoxic damage caused by the inflammatory responses.

How does all this information help us college students then? It doesn’t, really. This post merely serves as a warning that if we willingly pull all-nighters, we are setting ourselves up for a domino effect of immune responses and the homeostatic pressures exerted by our bodies in response. That, and cardiovascular disease. So, yes, technically, it really isn’t our fault that we’re not able to “heal” enough and in time to hand in our overdue assignments in time for their extended deadlines. But at the end of the day, who told us to pull an all-nighter (let alone 3) to begin with?

___________________________________________________________________________

Primary Reference:

Faraut, B., et al. (2011). Benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction. Brain. Behavior, and Immunity. 25:16-24.

Secondary References:

1) Takahasi, M., Arito, H. (2000) Maintenance of alertness and performance by a brief nap after lunch under prior sleep deficit. Sleep. 23:813-819.

2) Meier-Ewert, et al. (2004) Effect of sleep loss on C-reactive protein, an inflammatory marker of cardiovascular risk. J. Am. Coll. Cardiol. 43:678-683.

3) Redwine, L., et al (2010) Effects of Sleep and Sleep Deprivation on Interleukin-6, Growth Hormone, Cortisol, and Melatonin Levels in Humans. J Clin Endocrinol Metab. 85:3597–3603.

4) Irwin M.R., et al. (2010) Sleep loss activates cellular markers of inflammation. Arch. Intern. Med. 166;1756-1762.

5) Puttonen, S., et al. (2010) Shift work and cardiovascular disease—pathways from circadian stress to mortality. Scand. J. Work Environ. Health. 36:96-108.

6) Naska, A., et al. (2007) Siesta in healthy adults and coronary mortality in the general population. Arch. Intern. Med. 167:296-301.

7) van Mark, A., et al. (2010) The impact of shift work induced chronic circadian disruption on IL-6 and TNF-a immune responses. J. Occu. Med. And Toxicology. 5:18-22.

Thanks for a great semester!

Thank you all for your readership this semester. After over 8,000 page views and 850 comments, I hope that you've all learned a little bit about some new research in immunology! Student posts are wrapping up, but I hope to continue to post on at least a semi-regular basis going forth.  We'll be transitioning to the "Colgate Microbiology and Immunology Blog" henceforth, so look for some more student posts in the future as I offer other courses in Virology and Microbiology. In the meantime, here are some other blogs that I enjoy, some mainly immunology, others more general interest science. Thanks again, and check back with us as I continue to post!

- Geoff

Escaping Anergy

The Immunologist

Not Exactly Rocket Science

Macrophages: The Future of Easy Weight-Loss?

Think back to your first ever biology class on mammals. You most likely learned that all mammals, including humans, are warm-blooded (a.k.a., homeotherms) which means that our bodies remain at a constant temperature despite the temperature changes in our environment. So what is it that allows us to maintain this constant body temperature? Answer: thermogenesis.

Thermogenesis, essentially, is the conversion of stored fat into heat (1). We are most familiar with thermogenesis as shivering, which is the (inefficient) conversion of chemical ATP energy into kinetic, then heat energy. For this paper, however, Nguyen, et al. were most interested in non-shivering thermogenesis. The most recent model of thermogenesis dictates that when the hypothalamus senses cold temperatures, it triggers sympathetic discharge, releasing the hormone, noradrenaline, in brown adipose tissue (BAT), and white adipose tissue (WAT) (Fig.1).

[ Fig.1 ]

Nguyen, et al. found that when mice were exposed to cold temperature, both BAT and WAT activated adipose tissue macrophages that secrete catecholamines, hormones that trigger the fight-or-flight response. However, the effects downstream of catecholamines secretion differ between the two tissue types: catecholamines induce lipolysis in WAT, while in BAT, they induce thermogenic gene expression (4).

Macrophages are like the little ninjas of our immune system—they engulf and kill intracellular pathogen and pathogen components without any particular loyalty to a specific signaling pathway. Given this, they can be activated through two separate pathways: the creatively named classical and alternative activation pathways (3). Our classically activated macrophages, as per their common association, are pro-inflammatory and anti-parasitic in the classical pathway, with the inflammatory antiviral cytokine, IFN-g giving it orders The alternatively activated macrophage, on the other hand, is anti-inflammatory, and pro-survival (Fig. 2). Additionally, alternatively activated macrophages can facilitate wound healing. This pathway does not require priming, like the classical pathway, but often takes its orders from stimuli such as IL-4 and IL-13 (2).

Prion Diseases: Are Researchers Finally Close to Uncovering a Cure?

Prion diseases are no laughing matter, although it might have seemed that way if you were a visitor on the island of Papua New Guinea in the 1950’s. It was here that members of the Fore tribe were suffering from what they had dubbed the “laughing sickness” due to the strange uncontrollable bursts of laughter that accompanied the debilitating shivering that struck it’s victims. The source of the disease, which came to be known as kuru, was found to be transmitted through ingestion of brain tissue during cannibalistic burial ceremonies. Ingestion of the diseased victims brain tissues exposed tribe members to a structurally altered form of the normally expressed cellular prion protein (PrP) which could react with healthy forms of the proteins in their brains, perpetuating further structural transformations.

There are a number of diseases spread in this manner, termed transmissible spongiform encephalopathies (TSE). All TSE's, which include the well-known mad cow disease (Creutzfeldt-Jakob disease), are inevitably fatal and lead to neurodegeneration, often mediated through pathologic neuroinflammation. The inflammation seems to be primarily due to the robust activation of microglial cells (Yang et al., 2008). Microglial cells are akin to the brain’s garbage trucks, constantly collecting cellular debris from the extracellular environment. This behavior coincidentally makes them especially fit for detecting the presence of extracellular pathogens as well.

Before a cure can be developed, researchers must first uncover the cellular mechanisms underlying the microglial-medated pathologic neuroinflammation observed in the victims of TSE. PrP106-126 is the region of the PrP protein that has been shown to mediate inflammatory and pathologic signaling following structural alteration. A recent study was able to use this peptide to identify many of the proteins and signaling molecules, thus potentially uncovering future targets for pharmaceutical therapies.

You Are What You Eat: A New Therapeutic Approach to IBD

Virulent fimbria expression on bacterial mats in the absence and presence of Phloretin (Lee et al., 2011)

Many inflammatory bowel diseases (IBD) such as Chron's (CD) or ulcerative colitis (UC) are considered autoimmune diseases caused by the body's immune system attacking the gastrointestinal tract. This is thought to trigger colitis, the medical term used to describe colonic and often intestinal inflammation. Specifically, a failure to regulate T cell responses are believed to trigger the inflammation of the intestinal or colonic mucosa in these disorders.

Researchers have begun to suspect intestinal microflora inhabiting the mucosae play a significant role in the development of IBD and colitis. Patients suffering from IBD have been shown to have higher concentrations of mucosal bacteria that increase progressively with symptom severity. The mucosae are thin membranes that are found throughout the body in areas where internal organs and tissues are exposed to external environments. They possess unique immune systems that are tightly regulated and hyporesponsive, or unresponsive to most antigens, due to the high antigen loads they come into contact with (Neurath et al., 2007). Because the mucosae are so sensitive to inflammatory damage, disruption of the hyporesponsive state can have devestating effects.

Phloretin is a type of flavonoid found mainly in apples and strawberries that has antioxidative and anticarcinogenic properties, as well as biological roles in estrogen hormonal activity and cardiovascular disease prevention. If the roles of phloretin weren't already diverse enough, researchers have also recently found flavonoids can inhibit the biofilm formation of pathogenic E. coli O157:H7. Bacterial biofilms are aggregates of bacteria that aggregate together and adhere to a surface. This might explain another recent study that found phloretin is capable of reducing the expression of many inflammatory proteins and receptors in human colon epithelial cells.

Recently, this relationship between phloretin and biofilm formation was further investigated by a team of researchers hoping interested in whether phloretin's inhibitory effect on biofilm formation could have a therapeutic effect on patients of IBD (Lee et al., 2011).

CD147: The Maverick Triggering the Pathogenesis of Rheumatoid Arthritis

Pictorial representation of RA (http://www.rumatory-arthritis.com/)

Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disorder that leads to the synovial inflammation of joints and surrounding tissues. The clinical prognosis is the irreparable destruction of cartilage, tendon, and bone in affected joints and the disease can progress aggressively, especially without proper treatment.

The source of symptoms is thought to be the aberrant proliferation of a resident joint cell population, termed the synovial cells. The increased proliferation of these cells leads to the formation of an aggressive tissue body called the rheumatoid pannus, which expands into the cartilage and erodes the bone, producing the painful and debilitating effects characteristic of RA. CD147, the extracellular matrix metalloproteinase inducer (EMMPRIN) protein, is expressed at abnormally high levels in the synovial tissues of RA patients and is believed to increase matrix metalloproteinase (MMP) expression by synovial cells. MMP's target and break down a variety of proteins present in joint connective tissues, thus leading to the destruction seen in RA patients (Chen, 2004). Thus, MMP protein levels are thought to reflect disease activity an progression in RA better than levels of other inflammatory signaling molecules (Mahmoud et al., 2005). CD147's ability to increase the expression of MMP’s suggests it could play an important role in RA pathogenesis.

The destructive effects of abnormally CD147 expression may mediate other pathologic features associated with RA as well. Chronic inflammation in these tissues is maintained by angiogenesis, the growth of new blood vessels, through inflammatory cell transport to the synovitis and nutrient cell delivery to the growing pannus. Agiogenic factors thought to be involved in this positive regulation of angiogenesis include vascular endothelial growth factor (VEGF), a protein that triggers growth of blood vessel tissues, and hypoxia inducible factor-1α (HIF-1α), a protein that mediates cellular adaptation to sub-optimal oxygen conditions. Expression of CD147 was found to be positively correlated with VEGF and HIF-1α expression in the synovium of RA patients and experimentally mediated inhibition of CD147 signaling was found to reduce their production significantly. This led researchers to hypothesize CD47 could play an important role in the abnormal angiogenesis observed in the synovium of RA patients, and could promote pannus formation through enhancing local VEGF and HIF-1α expression (Wang et al., 2011).

Infect Me With Parasites? TGIT(GFbeta)

The use of helminthic therapy (the intentional infection of a patient with parasitic worms) to treat autoimmune diseases has enjoyed recent popularity for treatment of conditions as diverse as Crohn’s disease (1) and multiple sclerosis (2) . The jury is still out on its efficacy, however, and the thought of intentionally infecting patients with parasites for therapeutic purposes seems counterintuitive if not downright crazy. Part of the controversy stems from the conflicting reports as to exactly how helminthic therapy works. Some researchers suggest that helminthic therapy works by an evasive maneuver on the part of the parasitic worms to trick the immune system into producing the wrong kind of response, a type 2 cytokine response. (3) The authors of a recent study used helminthic therapy in a mouse model of non-obese diabetes (an autoimmune disorder) in order to attempt an answer to this question, which will be the focus of this blog post. (4)

The authors first demonstrated, by immunostaining, that mice deficient in IL-4 (a immune signaling molecule critical to the development of a type 2 response) fail to develop a type 2 response when infected with a parasitic worm in contrast to control mice that had normal levels of IL-4 which then developed a type 2 response. Given the role of IL-4 in promoting a type 2 response, this result seems fairly obvious and unnecessary to report. This is vital piece of data, however for the next experiment that these scientists performed.

The scientists then measured the onset of diabetes in the mice they were experimenting upon (remember they are specially designed to be genetically pre-disposed to develop diabetes) by measuring glucose levels in their blood. Surprisingly, mice infected with the parasite, regardless of competency in producing IL-4, did not develop diabetes. This suggests that a shift to a type 2 response is not the critical factor in the protection afforded by helminthic therapy in this model. What then, one might ask, are those wriggly worms doing to fend off diabetes?

The scientists who authored this study thought that it might have to do with regulatory T cells at first, but flow cytometry experiments they performed showed no difference in the numbers of regulatory T cells in any condition, infected/uninfected or IL-4 competent or not. They then hypothesized that either IL-10 or TGFβ, which have been implicated in regulating diabetes in mice could play a role. (5) Further flow cytometry experiments showed that TGFβ production was the critical factor in infected mice that correlated with the resistance to diabetes.

The results of this study suggest that the protection against diabetes afforded by helminthic therapy is not a result of a shift to a type 2 cytokine response, nor by regulatory T cells, but by an increase in the production of TGFβ, which is an immune signaling molecule that serves to tamp down the immune response. This opens up questions as to whether or not infection with parasitic worms is actually needed in order to convey the same protection that administration of TGFβ may be able to provide. This alternative to helminthic therapy must be explored further.

References

1) Summers, R. W., Elliott, D. E., Urban, J. F., Thompson, R., & Weinstock, J. V. (2005). Trichuris suis therapy in Crohn’s disease. Gut, 54(1), 87-90.

2) Benzel, F., Erdur, H., Kohler, S., Frentsch, M., Thiel, A., Harms, L., Wandinger, K.-P., et al. (2011). Immune monitoring of Trichuris suis egg therapy in multiple sclerosis patients. Journal of helminthology, 1-9.

3) Hübner, M. P., Stocker, J. T., & Mitre, E. (2009). Inhibition of type 1 diabetes in filaria-infected non-obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3+ regulatory T cells. Immunology, 127(4), 512-22.

4) Hubner, M. P., Shi, Y., Torrero, M. N., Mueller, E., Larson, D., Soloviova, K., Gondorf, F., et al. (2011). Helminth Protection against Autoimmune Diabetes in Nonobese Diabetic Mice Is Independent of a Type 2 Immune Shift and Requires TGF- . The Journal of Immunology.

5) Hancock, W. W., Polanski, M., Zhang, J., Blogg, N., & Weiner, H. L. (1995). Suppression of insulitis in non-obese diabetic (NOD) mice by oral insulin administration is associated with selective expression of interleukin-4 and -10, transforming growth factor-beta, and prostaglandin-E. The American journal of pathology, 147(5), 1193-9.

Asthma and Food Allergy

Asthma is a chronic inflammatory disorder afflicting the lower respiratory tract, characterized by a narrowing of the air passages. Roughly one in ten American children has asthma, and an estimated 6.5 million children under the age of 18 have been diagnosed with the condition. The prevalence of asthma is increasing, particularly amongst children, and the World Health Organization (WHO) estimates that each year 15 million disability-adjusted life-years are lost, and approximately 250,000 asthma deaths are reported [2]. Similarly, food allergy affects around 5.9 million children in the US, or 8% of children under 18 years of age. Moreover, nearly 2 out of every 5 children with food allergy have a history of severe reactions. In these cases, accidental exposure to an allergenic substance may result in breathing difficulties, an abrupt drop in blood pressure, and even death [3]. Both asthma and food allergies have serious implications, particularly amongst pediatric victims, and in a significant number of children, the two conditions are associated. Diagnosis with both asthma and food allergy presents a risk factor for fatal anaphylactic reactions.

Asthma and food allergy present examples of Type I Hypersensitivity (type I HS), in which an individual’s immune system incorrectly recognizes substances that are normally harmless as threatening invaders. As a result, an immune response is inappropriately mounted against them. The triggers for type I HS are referred to as allergens because they are antigens that cause allergy. Type I hypersensitivity reactions occur rapidly following the introduction of allergen in the body, and a particular subset of antibodies, called IgE, become implicated in the pathology of the immune response. These IgE antibodies, in cases of allergy, are directed against the allergen particles (including components of pollen, pet dander, or nuts, for example). During an asthma attack, inhalation of allergy-inducing substances or environmental triggers causes inflammation of the air passages, such that airways become constricted and breathing becomes quite difficult. In an allergic reaction to a food allergen (i.e. peanuts, soy, milk, eggs, wheat, or fish), a variety of possible symptoms may manifest, depending on whether the inflammatory response is induced in the mouth, the gastrointestinal tract, or enters the blood stream becoming systemic or provoking symptoms in a separate location of the body. In both asthma and food allergy, the exposure to allergen stimulates mast cells, a type of cell involved in inflammatory processes, to release the contents of their granules (cellular vesicles that contain substances like histamine, serotonin, and proteases). These fast-acting mediators are responsible for the rapid onset of initial symptoms associated with type I hypersensitivities because they are preformed within the cell, ready to be released upon activation. Histamine promotes blood vessel dilation and permeability, or leakiness, mucus production, itching, sneezing, and contraction of bronchial smooth muscle. Similarly, serotonin is associated with vasodilation and bronchial smooth muscle contraction. Proteases released by the mast cells are also involved in mucus production, as well as in increased blood pressure.

Approximately 4-6 hours following onset of these reactions, various white blood cells (leukocytes) migrate to the allergen-laden tissue. Especially significant in this stage of allergic reaction are eosinophils, which possess receptors on their surfaces that bind to IgE antibodies coating the allergen. Upon interaction of these receptors with the IgE-allergen complexes, the eosinophils undergo degranulation, similarly to the mast cells described above. The contents released from the eosinophil granules include leukotrienes, platelet-activating factor (PAF), major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin; when these substances diffuse into the surrounding tissue, they cause substantial damage to cells. Because the cells that line the airways exhibit elevated sensitivity to these mediators, symptoms of asthma are attributed predominantly to eosinophil activation during allergic reaction. This pathology is known as eosinophilic airway inflammation and is considered a distinguishing feature of asthma. Furthermore, worsening airway inflammation in pediatric cases of asthma is correlated with exacerbating symptoms. While asthma severity is known to be associated with eosinophilic airway inflammation, as well as with a co-diagnosis of food allergy, it had not been determined whether this type of inflammation is exacerbated in asthmatic children with food allergies, until quite recently. A study published in Pediatric Allergy and Immunology examined the hypothesis that eosinophilic airway inflammation is higher in children with both food allergies and asthma [1].

A Novel Role for Controlling GI Tract Bacteria

The gastrointestinal (GI) tract is covered with harmless bacteria, bacteria which are somehow able to evade the immune system cells that heavily populate this area. These bacteria are commensal, meaning they that both the bacteria and the organism they reside in are able to benefit from their presence in the gut. It has previously been shown that children who have increased diversity of bacteria in their GI tract during infancy are less prone to developing allergies when they reach school age ¹. It is also known that these bacteria play an important role in helping with the digestion of food, providing vitamin K, helping protect the colon from the invasion of harmful bacteria, and helping to educate the immune system to differentiate between harmless and harmful bacteria². However, the mechanism employed by these bacteria that allows them to reside in the GI tract without being attacked by the hosts immune cells is not well understood.

One type of cell found in the immune system is the B cell, which secretes antibodies, or proteins that neutralize an infectious pathogen, stopping it from harming the host. After these cells encounter a pathogen, they mature into plasma cells and go through isotype switching, a process that allows the cells to change the type of antibody they secrete. Before this switch occurs, B cells secrete an antibody called IgG, but after the switch, they start creating an antibody that specifically targets the type of pathogen that they came in contact with. It is already known that plasma cells in the GI tract secrete the antibody IgA, but it is unknown what supports this class switching and its role in facilitating homeostatic balance between the bacteria in the gut and the host's immune system.

Jorg H. Fritz and associates found that nitric oxide (NO), which is produced by inducible nitric oxide synthase (iNOS) was necessary for the isotype switching of B-cells in the GI tract to IgA producing plasma cells. They had already found that mice who lack lymphotoxin, a chemical that activates the production of the iNOS, also lack IgA. So, they hypothesized that iNOS may cause the B-cell to isotype switch to IgA secreting plasma cells.

Hope for an HIV Vaccine

In 1984, the Health and Human Services Secretary declared that extensive research for a vaccine protecting against Human Immunodeficiency Virus, a virus that ravishes the immune system of its host, would be available in two years. Almost three decades and one billion dollars later, scientists have still not been able to develop a vaccine that can successfully protect against the virus. The reasoning for these difficulties revolve around HIV's ability to rapidly mutate, making it near impossible for the immune system to mount an effective response (class notes, 12/8/2011).

During a normal immune response, an immune system cell called a B-cell makes antibodies, a protein that neutralizes specific proteins on a pathogen called antigens, preventing it from infecting the host. When a person gets vaccinated against a pathogen, their immune system is being given a form of the pathogen that does not have the ability to infect the host. This form of the pathogen allows the host's immune system to prime itself against the virus, which includes the creation of antibodies, so that when the host comes in contact with the infectious form of the pathogen, the antibodies will be waiting ready to stop the it.

However, the problem with HIV is not that it stops the body from making these antigens, but that it mutates so quickly that it is able to circumvent the antibodies. When an antibody is made, it will bind to a specific part of the outside of the pathogen, but once antibodies against HIV are made, the virus has already changed its external characteristics such that the antibody cannot bind to the virus and neutralize it. This is the main reason why making a vaccine against HIV has been so difficult.

Instead of injecting mice with parts of the virus in hopes of causing the creation of antibodies against HIV, Alejandro Balazs used a technique called vectored immunoprophylaxis (VIP). This involves inserting DNA for an HIV antibody into a virus known to be harmless to humans, called adeno-associated virus (AAV), and then injecting this virus into the host's muscle, in the hopes that it will cause the host to make antibodies that will neutralize HIV. These scientists injected VIP into mice and found that the mice were fully protected against the HIV virus.

New EAE Models More Accurately Reflect MS

Multiple Sclerosis (MS) affects about 400,000 people in the United States. In MS, the myelin sheath that coats our neuronal axons is degraded, as are the cells that produce myelin (also known as oligodendrocytes). MS is considered an autoimmune disease because the attack is facilitated by our body’s own immune system. This degradation of the myelin sheath affects the ability of our neurons to transmit electrical signals to each other. This manifests itself in the symptoms often associated with MS: numbness in limbs, paralysis and vision impairment.

In order to study MS, researchers often employ the use of animal models. Specifically, Experimental Autoimmune Encephalomyelitis (EAE) is a well recognized mice model that mimics the progression of MS. EAE is considered a Th1 focused disease with T cells secreting primarily IFNϒ. T cells are immune cells in the body that participate in cell-mediated killing of foreign pathogens (1). In MS, they recognize our myelin as a foreign substance and proceed to destroy it. One way they do this is by secreting cytotoxic cytokines, such as the aforementioned IFNϒ. When inducing EAE in mice, this Th1 response is ensured by injecting a myelin peptide(to mount an immune response against) along with complete Freunds adjuvant (CFA), which contains a bacterium called M. Tuberculosis (CFA).

Aside from IFNϒ, IL-23 has emerged as a notable cytokine because mice deficient for it remained protected against EAE pathology. Furthermore, IL-23 promotes the differentiation of inflammatory Th17 cells (2). Numerous EAE models currently exist; some more representative of MS in certain clinical regards (e.g., onset, clinical progression, and remission). Therefore, it’s vital to always explore new EAE models in an effort to find one that best represents human MS. In a recent study by Smith et al. 2011, researchers replaced M. tuberculosis with C. rodentium (CRA)in the injected adjuvant. CRA is a bacteria known to induce an IL-23 dependent Th17 response (as opposed to the aforementioned M.tuberculosis-mediated Th1 response) to find out whether different EAE phenotypes would emerge.

Natural Killing and Adaptive Immunity: the role of NK cells in CD8+ T cell differentiation

In the human immune response, an encounter with a pathogen results in the rapid division of immune cells which neutralize and kill viruses, bacteria and parasites. While this rapid increase in the number of immune cells in the body is necessary for a robust and effective immune response, once the pathogen has been cleared it is essential that immune cell levels be brought back down to normal. CD8⁺ T cells, also known as cytotoxic T lymphocytes (CTLs), are one type of immune cell that follows this pattern of expansion and contraction. In the presence of an antigen, CD8⁺ T cells specific for that antigen divide rapidly, seeking out and killing infected cells and pathogens. When the threat has been cleared, high levels of CD8⁺ T cells specific for a single antigen are no longer necessary, and the majority of CD8⁺ T cells die by apoptosis. The surviving CD8⁺ T cells differentiate into memory T cells, allowing the body to respond more quickly and effectively the next time it encounters the same antigen. This process is essential for maintaining immunity to diseases; the memory T cells and B cells produced after an initial exposure are the reason that, for example, you only get chicken pox once. Similarly, memory T cells and B cells enable the body to prevent infection by pathogens against which it has been vaccinated.

In a paper published in the Journal of Immunology just this week, Soderquest, et al. examine the relationship between CD8+ s and natural killer cells (NK cells) after an infection has been cleared. NK cells are an important part of innate immunity – that is, the rapid and general immune response that includes both physical barriers to infection (i.e. the skin and mucosa) and non-specific killing cells like the NK cell. During the innate immune response, NK cells identify and kill cells that are either infected with viruses or are tumorigenic. Apart from their role in the innate immune response, recent studies have shown that NK cells play an important part in the development of the more specific adaptive immune response, particularly in promoting the differentiation of CD4⁺ “helper” T cells (Th cells). In investigating the relationship between CD8⁺ T cells and NK cells, Soderquest et al. focused on the NKG2D pathway of NK cell killing. NKG2D is a protein expressed on the surface of NK cells that binds to NKG2D ligand (NKG2DL) on a target cell and induces apoptosis via the perforin/granzyme pathway. The authors of this article hypothesized that NK cells influence the development of CD8⁺ T cell-mediated adaptive immunity by killing activated CD8⁺ T cells via the NKG2D/perforin pathway.

Stopping Autoimmunity at its Roots: New Advances in the Treatment of Lupus Nephritis

Autoimmune diseases are the result of our own immune systems turning against us. There are various mechanisms through which autoimmunity can develop, most of which involve the breakdown in peripheral tolerance, which are the mechanisms our body puts in place to keep autotreactive T and B cells from damaging self tissue. If an autoreactive lymphocyte escapes central tolerance and finds its way to the periphery, it becomes the job of regulatory T cells (Treg cells) or tolerogenic DCs to anergize or delete the autoreactive lymphocyte. If there are abnormalities in regulatory T cells, then peripheral tolerance is hindered and an autoimmune disease could develop. Other conditions could result if problems exist in compliment deposition since C3b is responsible for helping immune complexes remain soluble when they pass through narrow channels in the body’s periphery. When cells are destroyed during an autoimmune attack, internal cell contents can be leaked and then work as antigens for the activation of additional lymphocytes. This occurrence may perpetuate an autoimmune response. Regardless of the mechanism, these responses are damaging to the host and require the development of effective treatments.

One damaging autoimmune disease, systemic lupus erythematosus (SLE), is caused by the production of “antinuclear” antibodies which target internal cell components such as DNA when these molecules are released from cells. This disease can affect the skin, joints, kidney, lung, heart, and brain. Since SLE’s symptoms are often varied, the disease can be mistaken for other illnesses. The mechanism of action of SLE has been linked to abnormal B cell development and activation. These B cells are also more sensitive to cytokines than would normally be expected. Furthermore, the fact that an increase in IL-10, a B-cell stimulating molecule, has been associated with SLE patients provides additional evidence that this disease is caused by B cells. This observation is interesting because typically we associate IL-10 as an immunosuppressive cytokine, however in the case of SLE patients, the immunostimulatory effects of IL-10 on B cells appear to outweigh its immunosuppressive value (1). SLE is considered to be a type-III hypersensitivity because these activated B cells produce autoantibodies that can form insoluble immune complexes that basically “clog up” narrow capillaries or other parts of the body such as the glomerulus, a spherical structure in the kidneys which filters blood. As a result, many SLE patients manifest the serious disorder called lupus nephritis. Lupus nephritis is a major cause of morbidity and mortality among SLE patients (2). It results when immune complexes interfere or cause damage to structures in the kidney, such as the glomerulus, and can rapidly worsen to kidney failure. Treatment for lupus nephritis typically focuses on the use of medications to suppress the immune system in order to improve kidney function. Dialysis, to control symptoms of kidney failure, and kidney transplantation are other treatments that may be recommended.

Recent research by K. Ichinose and several colleagues at Beth Israel Deaconess Medical Center and Harvard Medical School has been focusing on the cause of lupus nephritis rather than on new treatments for the malady. The researchers are hoping that their efforts will lead to the development of a more targeted drug which can do more for patients than the current drugs that work by suppressing the immune system on a large scale. They chose to study mesangial cells (MC’s) in the glomerulus because they proliferate during lupus nephritis, a phenomenon that could link MC’s to the cause of this autoimmune disease. Typically, the function of these specialized cells is related to support, filtration, and phagocytosis of immunoglobulin. These cells can also produce the proinflammatory cytokine IL (interleukin)-6, found during glomerular inflammation. The researchers also looked at calcium/calmodulin-dependent kinase type IV (CaMKIV). This kinase belongs to a family of kinases that regulates autoimmunity and cell proliferation. CaMKIV is a multifunctional protein that is highly expressed in the central nervous system. Because increased expression of CaMKIV has been linked to certain cancers, some researchers see this as evidence that it is involved in cell proliferation (3). This observation led them to perform tests to ascertain whether CaMKIV could be deleted or its actions blocked, possibly leading to decreased MC proliferation and IL-6 production that could in theory alleviate an autoimmune response.

Helpful Hormones: The potential benefits of hormonal contraceptives on delaying HIV progression to AIDS

Any person who has sat through “sex-ed” has heard about practicing safe sex, using condoms to prevent the transfer of STDs from one partner to another. Additionally, there are other methods of contraceptives, namely hormonal contraceptives. Once one has contracted HIV there is an even increased necessity to protect against pregnancy in order to prevent transmission of disease to the child and to protect the mother against unwanted pregnancy. However, with hormone treatments there have been studies that indicated the hormonal contraceptives actually advance the progression of HIV in patients.¹ While this has mainly been documented in undeveloped countries, any implication in the advancement of HIV towards AIDS is a concern and should be studied.

Some of the largest populations of HIV positive persons are the commercial sex workers and the populations in sub-Saharan Africa.² One study in particular followed a group of HIV seroconverted women located in Uganda for a person of 11 years. This was one of the most extensive studies with one of the largest sample sizes, thus increasing the possibility for significant results (625 women in study). The women in this study were compared for differences between the hormonal contraceptive users and nonusers and monitored for the change from HIV to AIDS or death. Hormonal contraceptives in this study were considered both oral contraceptive pills and implanted hormonal contraceptives.

In the entire study 27% of participants used hormonal contraception at some point during the study. What the researchers found was that, in general, women who reported using hormonal contraceptives had a longer delay to onset of AIDS and a longer time until death. Specifically, those who used hormonal contraception had on average 3.92 years until death, and 3.72 years until AIDS, compared to 3.04 years until death and 2.98 years until AIDS or those without hormonal contraceptive use. Interestingly, this study also took note as to what kind of behavioral patterns the hormonal contraceptive users engaged in compared to the nonusers. This study found that women using hormonal contraceptives had on average a higher degree of education, were in the median age category (25-35), and were also less likely to use condoms. These have to be taken into account as well as the hormonal contraceptive use as possible variables affecting onset of AIDS and death.

While there have been studies that have linked hormonal contraceptives with an increase in time to onset of AIDS, the results of this study are not unheard of. Some previous studies have suggested that the hormonal contraceptives may actually reduce HIV progression.³ This study was also quick to point out their strengths, namely that their study provides evidence that hormonal contraceptives do not have an impact on the progression of HIV to AIDS/death. However, there were also limitations that this study had to deal with and should be pointed out in this discussion. In particular, it was very difficult to determine a death as AIDS related as opposed to outside causes. Another problem with this study was the difficulty in determining the varying affects that differing hormonal contraceptives had on varying the degrees of HIV progression.

The big picture to take away from this study is the vast array of treatments and possible drug therapies that can be applied to HIV/AIDS patients. There are large groups of people worldwide who are HIV seroconverters that are being observed and studied in order to advance preventive technologies. It is important that all possible treatments are considered as there has yet to be an effective vaccine developed. While the specific biological interactions were not considered in this study, there is still a future direction in hormone treatment, and anything that can slow the onset of AIDS is worth a second look.

Primary Resource

Polis C, Wawer M, Kiwanuka N, Laeyendecker O, Kagaayi J, Lutalo T, Nalugoda F, Kigozi G, Serwadda D, and Gray R. (2010). Effect of hormonal contraceptive use on HIV progression in female HIV seroconverters in Rakai, Uganda. AIDS. 24:1937-1944. Doi: 10.1097/QAD.0b013e32833b3282

Additional Resources

1. Stringer EM, Kaseba C Levy J, Sinkala M, Goldenber RL, Chi BH, et al. (2007) A randomized trial of the intrauterine contraceptive device vs hormonal contraception in women who are infected with the human immunodeficiency virus. Am J Obstet Gynecol 197: 144-148

2. “UNAIDS: Sub-Saharan Africa” WHO. 2009 091124_fs_ssa_en.pdf>

3. Allen S, Stephenson R, Weiss H, Karita E, Priddy R, Fuller L, et a. (2007) Pregnancy, hormonal contraceptive use, and HIV-related death in Rwanda. J Womens Health. 15:1017-1027

A New Treatment Option for Alzheimer's Disease? IL Take It!

Alzheimer's disease (AD) is a devastating neurodegenerative disease that is, according to the National Institute on Aging, the most common form of dementia among older people. (1) Dementia is characterized by impairment in many mental faculties including, but not limited to: language, memory and perception. (2) Neuroinflammation is known, at this point in time, to play a role in AD. What exactly inflammation has to do with AD is currently a topic of much debate in scientific circles. An analysis of microarray data using microarray techniques identified 5 cytokines (immune system signals) to be important biomarkers of AD. (3) One of the cytokines identified in that study is IL-1, the production of which has been identified to be critical to the formation of the filamentous protein tangles that are a hallmark of AD. (4) This led a team of researchers at the University of California, Irvine to attempt inhibition of IL-1 signaling in a mouse model of AD with the application of an antibody directed against IL-1R (the receptor for IL-1) with the hope of improving AD symptoms. (5) This study is the focus of this blog post.

The researchers first did a behavioral study to test the efficacy of the antibody treatment on the cognitive abilities of their murine subjects. These mice are not your average laboratory mice; they have had three genes introduced into their cells that result in the accumulation of protein deposits and neuronal miscommunication that plague AD patients. (6) After treatment with the anit-IL-1R antibody, mice showed considerable improvement over non-treated mice in terms of their ability to navigate a water maze and to recall traumatic events (see figure below).

CD4+ T cells in HIV

Human immunodeficiency virus (HIV) is a member of the retrovirus family and progresses into AIDs after a period of time. AIDs is defined by a significant deficiency in T-cell count which causes a progressive failure of the immune system and allows life-threatening opportunistic infections and cancers to thrive. Infection with HIV occurs by the transfer of blood, semen, vaginal fluid, or breast milk. In order to slow the progression to AIDs, many people with HIV take antiretroviral therapy which combines a number of drugs that are designed to stop HIV from infecting cells: includes nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, and triple-nucleoside (or nucleotide) reverse transcriptase inhibitors (NRTIs) (1). HIV initially depletes the body’s CD4 T-cells and the point of this therapy is to boost the immune system back to normal to continue to fight the infection. However, 30% of patients with HIV who receive antiretroviral therapy fail to achieve a normal CD4⁺ T-cell count (2). This failure to achieve normal T-cell levels results in a steady decrease in T-cells to where, in less than 10 years, the count is below 500 (signifying AIDs) leaving the patient open to opportunistic infections and eventually death. Therefore it is necessary to study the reasons for this initial failure to reach normal T-cell counts with antiretroviral therapy in order to enhance its function.

HIV first infects CD4 T-cells, depleting the amount in the body. However, there is normally a period of time in which the T-cells regenerate in order to fight the infection. The balance of T-cells in the body is negatively regulated by interferon α (IFN-α) and evidence suggests that HIV increase the production of this interferon (3). This regulating interferon may promote apoptosis of uninfected CD4⁺ T cells by up-regulating expression of a death signal (TRAIL) and its death receptor (TRAIL receptor) (4). Therefore, the role of IFN-α may have important effects on CD4 T-cell populations in HIV patients.

A study by Sonia Fernandez at the School of Pathology and Laboratory Medicine in Australia looks at the effects of IFN-α on CD4 T-cell populations in HIV patients. They recruited HIV patients who have been receiving effective doses of antiretroviral therapy and measured the levels of CD 4 T-cells in the blood as well as levels of IFN-α. The patients were divided into low or high CD4 T-cell groups. Expression of markers of T-cell activation (HLA-DR), apoptotic potential (Fas), or aging (CD57) were assessed and it was found that the proportions of CD4⁺ T cells expressing HLA-DR or Fas were higher in patients with low CD4⁺ T-cell counts than in those with high counts such that the higher the levels of Fas and HLA-DR that are expressed, the lower the population of CD4+ T-cell count. The proportion of CD4⁺ T cells expressing CD57 did not differ between patients with low or high CD4⁺ T-cell counts. They left the cells in a culture for 72 hours and found that the proportions of CD4⁺ and CD8⁺ T cells that were apoptotic or preapoptotic was similar in patients with low and high CD4⁺ T-cell counts.