The Role of CD40–CD154 Interactions in Autoimmunity and the Benefit of Disrupting this Pathway

Autoimmune disease is associated with adaptive immune component dysfunction such as the B cells and T cells. The cells somehow pass through peripheral tolerance and cause a self reaction. Autoimmunity is determined by genetics, environmental, and hormonal effects that contribute to the disease. Many autoimmune diseases have been studied and examined but the biology behind them are not completely understood.

A major pathway is the CD40-CD40 ligand (CD154) because it is needed to activate many adaptive immune cells such as DC's, B cells, and T cells. This interaction has multiple functions and as costimulatory molecules, they are upregulated in several autoimmune diseases for example SLE (systemic lupus ertyhematosus). It was thought that disabling this interaction could be a new avenue of therapy for autoimmunity.

Caption: The image to the left shows the basic interactions between immune cells in the body when trying to get rid of an antigen. The same interactions occur in autoimmune diseases where the body is attacking self antigens. In this picture, we can see the CD40-CD40L (CD154) interaction described above between a dendritic cell presents antigens and a CD4+ T cell which aims to help the immune response by releasing cytokines.

CD40 provides a help signal to dendritic cells to maturation. The duration of the signal assists in determining the function of the dendritic cells which is displayed in this study. There are various other proteins that can trigger dendritic cells to become activated and thus spark an autoimmune response such as heat shock proteins. These can skew a response using cytokines, interleukins, and different factors. In a mouse model, diabetes was induced when CD40 expression on bone marrow was necessary for heat shock protein induced activation. 

This reaction of CD40-CD154 is not only found in dendritic cells but it also impacts T cells by priming them, polarizing T cells to a certain response.

Caption: The basic polarization of T cells is shown in the following image with different cytokines determining the Th cell developed. 

Th (T helper) cells only express CD154 after they have been stimulated by an antigen which allow them to have this interaction of CD40-CD154; unfortunately over expression of CD154 can lead to autoimmune diseases. This not only occurs in T cells but in B cells as well. The CD40-CD154 interaction enhances CD86 expression on B cells in autoimmunity specifically SLE disease which is a hypersensitive reaction. CD86 contributes to the presentation of self-antigens to T cells thus inducing a negative immune response by the body. 

Transglutaminase-2 and Celiac Disease

Celiac disease is an autoimmune disease that has become increasingly prevalent in recent years.   It is an immune reaction that occurs with gluten, which is a protein found in wheat, barley, and malt.  This reaction causes inflammation, which damages the small intestine.  You may be thinking that wheat, malt, and barley are in everything!  So, what do people who suffer from celiac eat?  As someone who suffers from celiac, I can tell you the transition to a gluten free diet wasn’t easy, but luckily there are a lot of good, gluten free alternatives that have been developed.  Even though celiac can be helped with dietary accommodation, it is still important to try to understand what is causes the autoimmune response in order to potentially develop a treatment for celiac disease someday.  Unfortunately, it is not well understood how the autoreactive B cells become activated in celiac, but it is known that celiac disease is associated with autoantibodies produced that are specific for the transglutaminase-2 (TG2) enzyme.  In the paper Transglutaminase 2-Specific Autoantibodies in Celiac Disease Target Clustered, N-Terminal Epitopes Not Displayed on the Surface of Cells, the authors Iversen et al. investigated the mechanism that controls the formation of the TG2 autoantibodies.

Transglutaminase-2 is an enzyme that is involved in the deamination of glutamine residues, during them into glutamic acid.  This process increases their affinity for the HLA molecules associated with the disease, thus increasing the reactivity of gluten.  The anti-TG2 autoantibodies are an important marker of celiac disease, but it is unknown how these autoantibodies are contributing the disease.  In the lab’s previous research, they have identified various monoclonal (target 1 particular antigen) TG2-reactive antibodies (mAbs), and they have found that the antibodies target distinct (but close or overlapping) regions.  They define these regions as epitopes 1-4 throughout the paper. 

           

In order to determine the specificity of the mAbs, they stained the small intestinal tissue sections with immunofluorescent dye in either wild-type or mice deficient in TG2.  There was only fluorescence (indicating mAb binding) in the tissues containing TG2, which suggests that the mAbs are highly specific to TG2.  They also attempted to determine whether the mAbs would react with other members of the transglutaminase family, TG3 and TG6, which have also been associated with celiac disease.  They did not find any reactivity, which indicated that autoantibodies against other transglutaminases in celiac disease are created in dependently than the anti-TG2 autoantibodies.

TG2 has both an open and closed conformation.  It was thought that the epitopes on TG2 targeted by the autoantibodies were conformational, it would be expected that the mAbs would differ in binding strength to the two conformations.  Using an ELISA assay, the authors used the natural conformational regulators of TG2, Ca²⁺ (open conformation) and GTP (closed conformation) to measure the binding strength of the mAbs in each of the conformations.  The binding affinity of the mAbs was increased by Ca²⁺, but decreased by GTP.  This suggests that in celiac disease the open conformation of TG2 is the one that is targeted by the autoantibodies.

In a recent study done by Simon-Vecsei et al. , they identified an epitope targeted by celiac disease serum autoantibodies.  The authors wanted to know if their mAbs would react with the newly discovered epitope.  They constructed a triple mutation in the epitope and measured the binding strength with the mAbs by ELISA.  These mutations resulted in loss in reactivity for the mAbs in the epitope 2 group and the epitope 3 group.  This suggests that while this epitope is one of the major targets of autoantibodies in celiac disease, it is not the only one since the mAbs in the epitope 1 and 4 groups do not interact with it. 

Immunosupression of Type 1 Diabetes

Physiological changes in T1D vs a healthy person
(inability to control glucose levels due to lack of insulin being produced)

Type 1 diabetes (T1D) is a serious, yet often overlooked, autoimmune disease that affects approximately 3 million Americans.  It is often lumped into the general category of “diabetes” alongside type 2 diabetes, even though T1D is generally more serious and isn’t preventable.  In people with T1D, islet, or beta (β) cells, in the pancreas are slowly destroyed by the body.  These cells are responsible for producing insulin, which controls blood glucose levels and keeps them at a healthy level.  People with T1D have to constantly monitor their blood sugar and inject themselves with insulin in order to control their glucose levels or risk serious complications such as blindness, foot amputation, or even coma.  As of now, there is no cure for the disease.  Recent research has focused on trying to find a way to prevent the body from attacking itself through manipulation of the immune system and/or to restore β cell function.  However, even an improvement in treatment would greatly increase the quality of life for T1D patients.

            

Creation of Insulin: proinsulin cleaved to
produce active insulin and C-peptide

Autoimmune diseases are more complex to treat since it’s often very difficult to suppress only the part of the immune system that isn’t functioning.   Similar to cancer, it’s hard to kill only the bad cells in the body without affecting the good ones.  Roep et al have taken a step in the direction of successful immunosupression of the bad immune cells involved with T1D.  In order to understand their work, it’s first necessary to understand a little more about insulin and the immune system.  Insulin isn’t synthesized in its functional state.  It is synthesized as pro-insulin and then cleaved into an active portion with the A and B peptides, which is insulin, and an inactive portion, called C-peptide; you can’t have one without the other (as seen in the figure to the left).  When doctors want to measure β-cell function, they look at C-peptide levels.  So the researchers use C-peptide levels to determine if their methods are preventing immune cells from destroying insulin-producing cells.

Innate Immune System may be more involved in Multiple Sclerosis than Previously Thought

        Among the many types of autoimmune disorders perhaps the most interesting and least understood disorders are those in which the immune system attacks the central nervous system.  Multiple Sclerosis (MS) is an autoimmune disorder in which invading lymphocytes attack the protective insulation of neurons called myelin.  The disease is neurodegenerative and leaves patients wheelchair bound within a few short decades of its clinical presentation.  While scientists have unraveled many of the pathological hallmarks of the disease, its cause and effective treatment remain elusive. 

Lesions in an MS brain

            A new study in the Journal of Immunology published this month investigates the types of white blood cells that are involved in the attack of white matter (myelin) in the brain.  Previous studies have identified a subset of Helper T cells as a major cause of the pro-inflammatory environment which leads to the ultimate degeneration which is characteristic of the disease.  These cells are part of the adaptive immune systems which targets very specific pathogens and in some cases (like in MS) accidentally targets cells or proteins that are part of the body’s normal function.  However, the new article presents data that demonstrates that innate immune cells called neutrophils may be playing a role in the initial stages of the disease.  These innate immune cells are non-specific to any particular protein but instead identify and attack cells with general molecular patterns associated with invading pathogens.  This is the first time it has been shown that neutrophils have been involved in MS.  

Keep the hygiene, lose the inflammation: TGF-β and helminthic therapy

            Incidences of autoimmune and autoinflammatory diseases, such as type 1 diabetes, rheumatoid arthritis, and inflammatory bowel disease, are increasing in the developed world, and the annual costs of treatment amount to billions of dollars in the United States alone (1). There are numerous factors that can account for this rise in prevalence, including genetic differences and environmental factors. One factor that has been gaining support, both correlatively and experimentally, is the hygiene hypothesis: basically, since we have eliminated many of the prominent childhood diseases, children are “too clean,” and thus their immune systems, rather than focusing on pathogens, are stimulated to attack “self” targets instead, or target innocuous factors leading to allergy. In other words, infection with some types of pathogens, particularly parasitic worms such as roundworms, flatworms, and hookworms, can dampen the inflammatory responses that underlie many of these autoimmune/autoinflammatory conditions. We have largely eliminated these infections, which are transmitted through unclean drinking water and soil, in developed nations, thus accounting for some of the rise in autoinflammatory diseases.

            Some suffering patients have taken this idea to the extreme, and have deliberately infected themselves with parasites to try to alleviate their symptoms. Although this treatment is highly experimental and there is not a lot of clinical data demonstrating efficacy, anecdotal evidence suggests that some patients have observed marked reduction in symptoms following “helminthic therapy.” Obviously, there are many problems associated with introducing parasitic organisms into people, so if scientists can determine how helminthic infection tamps down inflammatory responses, it could potentially lead to novel anti-inflammatory treatments that don’t involve the parasites themselves.

            At the core of this issue are two major categories of immune responses, named “Th1” and “Th2” responses for the types of helper T cells that facilitate them. Th1 responses are inflammatory in nature, and involve the activation of macrophages and killer (CD8+) T cells. These responses are elicited by the secreted cytokine interferon-γ (IFN-γ), and typically target intracellular pathogens such as viruses. Th2 responses are mediated largely by B cells, which make antibodies, and are elicited by the cytokine interleukin-4 (IL-4). Th2 responses target extracellular pathogens, such as parasitic worms, and the rationale behind the hygiene hypothesis is that by eliminating many of these Th2 pathogens, the immune system “skews” towards Th1 responses. This, then, leads to increased inflammation and the associated autoinflammatory diseases. However, other responses caused by helminth infection, including the generation of regulatory T cells (T_Regs) and the secretion of immunomodulatory cytokines such as IL-10 and transforming growth factor-β (TGF-β), might also mitigate autoinflammatory disease. A recent paper by Hübner et al attempted to distinguish which of these mechanisms was responsible for the protection from autoimmunity accorded by helminth infection. They found that generation of a Th2 response was not required, but that the production of TGF-β was largely responsible for protection.

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.

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).

Commensal Microorganisms in the Gut: Friend or Foe?

Before we leave a public bathroom, how many times do we see or hear the phrase, “Did you wash your hands?” Chances are, we have seen or heard this plenty of times. As children, we were often reminded by our parents to wash our hands before and after eating, after handling dirty items, or after going to the bathroom. The obvious reason is to eliminate the germs that we have come into contact with throughout the day. However, as we all know, bacteria are not entirely harmful to our well-being. In fact, some bacteria in our gut function to keep us healthy. They help digest food, aid us in acquiring vitamins and nutrients, train our immune system, and compete with pathogenic bacteria that may otherwise be harmful to us. As such, they are called commensal microorganisms. Certain diseases caused by pathogenic bacteria are treated by a process called fecal matter transplant. This medical procedure involves the introduction of the normal gut flora obtained from the stool of a healthy donor in order to restore the gut microbiota of the infected individual. The concept behind this procedure is that the commensal microorganisms obtained from the stool will eventually outcompete the pathogenic bacteria responsible for the disease. For example, Pseudomembranous colitis, which is caused by Clostridium difficile, uses fecal matter transplant as one feasible treatment option. In the process, non-pathogenic bacteria are introduced into the colon of the patient in order to outcompete C. difficile for resources. However, is the commensal microbiota as beneficial as it is portrayed? Studies aimed towards understanding the pathology of multiple sclerosis (MS) would beg to differ.

MS is an autoimmune disorder of the central nervous system (CNS) where the myelin sheath, which wraps around the axon and facilitates the conduction of nerve signaling, is attacked by the immune system. The resulting demyelination causes signals between neurons to be delayed and this consequence is manifested through clinical symptoms that include numbness, motor dysfunction, and vision problems. In terms of the immune response, MS is mostly associated with increased CD4+ T cell infiltration into the CNS. This T cell infiltration is also accompanied by increased production of autoantibodies by B cells, which can attack the specific myelin proteins that make up the myelin sheaths. Several studies are aimed towards understanding what goes awry in MS, however, numerous questions remain unanswered regarding this disorder. Microbial infection has been attributed to this autoimmune disorder (Ascherio et al. 2007). As mentioned earlier, the commensal microbiota has been linked to the treatment of certain diseases by outcompeting pathogenic microorganisms. Therefore, could the microbiota also play a role in treating MS? A recent study shows that in the case of MS, commensal microbiota triggered autoimmune demyelination instead of alleviating MS symptoms.

CaMKIV: A Potential Target for a Treatment for Lupus Nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disorder that currently has no cure (PubMed Health, 2011). SLE may result in abnormal deposits in kidney cells, which leads to complications in the kidneys. This results in the condition lupus nephritis, in which extensive damage to the kidney occurs and ultimately results in kidney failure (PubMed Health, 2011). There has been much speculation that Calcium/calmodulin-dependent protein kinases (CaMKs) is a factor in the progression of SLE, for it has previously been demonstrated that CaMKIV contributes to decreased IL-2 production in SLE T cells (Karin, 1995). CaMKIV may also play a role in the production of IL-6, for it has been demonstrated that just before the age when lupus nephritis is developed, spleen cells of mice contain a subpopulation of Th cells that selectively induced their B cells in vitro to produce highly cationic IgG autoantibodies to both single-stranded DNA and double-stranded DNA (Datta, 1987). This may be of critical importance, for it has also been shown that IL-6 is a feed forward process in which macrophages respond to IgG to support further B-cell production of IgG (Maeda, 2010).

It has previously been demonstrated that inhibiting CaMKIV in lupus-prone MRL/lpr mice mitigates disease pathology (Karin, 1995). Ichinose et al. have recently elaborated on the cellular abnormalities that may be controlled by CaMKIV (2011). These results have helped to clarify the cellular processes that result in the damage of kidneys in lupus nephritis by producing large amounts of autoimmune mesangial cells and increasing production of IL-6 and have shown that CaMKIV may be a critical target in developing treatments for lupus nephritis.