New Kid on the Block: Th17

Dr. Cohen briefly mentioned this newly docuemented subset of T cells in class the other day. I thought I'd follow up on his lecture and give a little more information on the "new kid on the block".

The discovery of Th17 subset came from studies of autoimmune diseases such as experiment autoimmune encephalitis (EAE) and collagen induced arthritis (CIA). Originally people thought these were disease of the Th1 subset. It is understood that dendritic cells produce IL-12, which preferentially selects a Th1 response. Thus, people thought that the autoimmune disease above were Th1 dependent because upon treating with antibodies against the p40 subunit of IL-12, development of EAE and CIA were eliminated. However, in 2000, some extremely clever people discovered that there is a new member of the IL-12 family which shares the common p40 subunit of IL-12. Briefly, most cytokines are made up of separate subunits. Within a class of cytokines, there is a mix and match of these subunits. Therefore, IL-23, the newly described member, utilizes the same p40 subunit, to which neutralizing antibodies eliminated EAE and CIA described above. Upon this discovery, there were a series of mouse experiments carried out where mice deficient in IL-12, IL-23, or both were checked for susceptibility to EAE and CIA. Interestingly, the disease did not develope in mice lacking IL-23, suggesting that EAE and CIA are diseases that are specific to a subset of T cells that, upon stimulated by IL-23, produces IL-17 (this is simplified, of course, a lot of work was done for people to come to this conclusion). Similar to Th1 and Th2, production of cytokines from these two groups block the development of Th17 and vice versa.

IFN-gamma and IL-4 produced by Th1 and Th2 respectively are inhibitory for Th17 differentiation. TGF-beta is an essential growth factor that is required for Th17 differentiation. Historically, TGF-beta is known to have anti-inflammatory activities. They are produced by regulatory T cells and are thought to inhibit Th1 and Th2 activities. Inhibition of Th1 and Th2 activities therefore leads to a lack of IFN-gamma and IL-4 production, allowing for the environment to be permissive for Th17 differentiation. TGF-beta has also shown to push regulatory T cell differentiation. However, these are two distinct subset of T cells. It is thought that differentiation to one of the other is mutually exclusive, therefore a T cell cannot be both a regulatory T cell and a Th17 T cell. In addition to TGF-beta, IL-6 is also essential for Th17 differentiation (and inhibitory for regulatory T cell differentiation) while IL-23 may be important in sustaining the Th17 population.

This subset of T cells is particularly interesting to me. Since TGF-beta is usually an anti-inflammatory molecule (by inhibiting Th1, Th2, and upregulating Treg differentiation), why then is it pro-inflammatory for Th17? In other words, what is the special role that Th17 play? As mentioned before, Th17 are associated with autoimmune diseases and chronic inflammation. If we can inhibit Th17, is that sufficient to completely get rid of the disease? What this subset of T cells has gotten me to wonder is whether or not we can actually group T cells in terms of their subsets, in other words, are there subtle difference between all T cells such that if we just look at the "right" markers, or the "right" cytokines then ALL T cells are in fact in a different subset?

Constantinescu, C.S., et al (1998). Antibodies against IL-12 prevent superantigen induced and spontaneous relpses of experimental autoimmune encephalomyelitis. J. Immunology. 161, 5097-5104.

Oppmann, B., et al (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23 with biological activities similar as well as distinct from IL-12. Immunity 13, 715-725.

Park, H., et al (2005). A distinct lineage of CD4 T cells regulates tissue inflammation by producing Interleukin 17. Nat. Immunology. 6, 1133-1141.

Mangan, P.R., et al (2006). Transforming growth facto-beta induces development of the Th17 lineage. Nature 441, 231-234.

Bettelli, E., et al (2006). Reciprocal developmental pathways for the generation of pathogen effector Th17 and regulatory T cells. Nature 441, 235-238.

NF-κB in Tumorigenesis, or not?

Inflammation has been implicated in tumor promotion and progression in a number of cancers. In many cases, tumorigenesis has been linked to chronic infection. For instance, HBV or HCV infection has been shown to be a major risk factor for hepatocellular carcinoma. Nuclear Factor kappaB (NF-κB) is known to play a key role in the response to infectious agents by stimulating the immune response through regulation of pro-inflammatory cytokine genes. In addition, NF-κB has been shown to regulate many genes that are implicated in cancer. Some of these genes include cell-cycle regulators, genes involved in apoptosis, and proteases which have been shown to increase metastases.

In prostate tumorigenesis, NF-κB has been shown to induce secretion of IL-1 from infiltrating macrophages. In prostate cells, IL-1 causes the conversion of androgen antagonists to agonists. Increased androgen activity in the prostate has been shown to promote tumor formation.

NF-B has also been shown to inhibit tumor progression in females. Kupffer cells (a type of macrophage) present in response to cellular damage in the liver, release IL-6 which acts as a pro-growth and inflammation signal in neighboring cells. Prolonged exposure to IL-6 in the liver has been correlated with cancer formation. In Kupffer cells, estrogen acts as a suppressor of IL-6 by activating NF-B which then down-regulates this cytokine. Therefore, pre-menopausal women that produce a fair amount of estrogen have a much lower risk of developing hepatocellular carcinoma. NF-B activation has also been shown to have antiapoptotic effects in gastric cancer cells, though the mechanism is not so clear.

NF-B can be expressed in just about every cell type and can be activated by a number of pathways. Each pathway has the ability to cause NF-κB to target different subsets of genes. The ability of different signals to induce NF-κB targeting toward pro-cancerous or anti-cancerous genes is an interesting observation and should be considered when therapeutics are designed. Most NF-κB related therapies which are currently in clinical trials, aim to inhibit its regulation, which could have profound effects on the recipients immune system. Instead of inhibiting NF-κB, should we instead try to change its targets from less favorable to more positive ones? One example is the ability of estrogen receptor (ER) to target genes through NF-κB. Estrogen receptor normally activates or represses genes by binding directly to target genes that contain estrogen response elements. When ER binds certain ligands, it can also bind to and activate NF-κB, which leads to activation of NF-κB target genes. Estrogens are thought to elicit some of their pro-inflammatory effects in this way. Is it possible that, if exploited, this ER-NF-κB interaction can lead to an overall positive outlook for cancer patients? Can some other pathway that leads to NF-κB activation give a positive outlook? Since NF-κB controls so many genes (good or bad depending on context), should it even be considered as a target for therapy?

1. NF-κB activation in development and progression of cancer. Jun-ichiro Inoue,1,2 Jin Gohda,1 Taishin Akiyama1 and Kentaro Semba1 Cancer Sci. March 2007. vol. 98. no. 3. 268–274

2. Cancer: an infernal triangle. Alberto Mantovani. Nature 448, 547 - 548 (2007).

3. Nuclear factor-B in cancer development and progression. Michael Karin1. Nature 441, 431-436 (25 May 2006) | doi:10.1038/nature04870; Published online 24 May 2006.

4. Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori. Maeda S, Yoshida H, Mitsuno Y, Hirata Y, Ogura K, Shiratori Y, Omata M.

Mol Pathol. 2002 Oct;55(5):286-93.

5. Nuclear factor kB as a target for new drug development in myeloid malignancies. Cilloni D, Martinelli G, Messa F, Baccarani M, Saglio G. Haematologica. 2007 Sep;92(9):1224-9. Epub 2007 Aug 1.

Which state is skinniest?

Congratulations to our neighbors to the southwest (Arizona, for the geographically challenged) on being the 9th skinniest state in the USA, according to the Trust for America's Health (data from the CDC).

Of course, the skinniest state of them all is...guess who?

C'mon, AZ, if everybody there loses a kilogram, you can leapfrog over Utah, Montana, Rhode Island, Connecticut, Hawai'i, Vermont, and Massachusetts, and rank second!!

(I posted this because obesity is an inflammatory condition, right?)

Trust for America's Health site

The role of T cells in Neuroinflammation

I found Neuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson's disease to be a breath of fresh air since it provided an example of a good immune response after reading so many articles about immune responses gone awry. This article provided a wealth of information on the roles of regulatory and effector T cells. What I found particularly interesting in this article is the fact that regulatory T cells (Tregs) provided an overwhelming neuroprotective role in the substantia nigra pars compacta as Reynolds et al reported 90% protection of the dopaminergic neurons in the nigrostriatal system. In contrast, when MPTP injected mice were treated with effector T cells (Teffs), there was little evidence (2-18%) of neuroprotection (1087). It seems as though part of the reason Tregs are so effective as neuroprotectors is that they appear to recruit other anti-inflammatory agents. The study reported "significantly higher levels of IL-10 and TGF-β in MPTP/Treg-injected mice...compared with any other MPTP-intoxicated group" (1086).

Reynolds, et al did a very thorough job of looking at the role of Tregs and Teffs in several cellular aspects of Parkinson’s disease. They found that Tregs attenuated evidence of neurodegeneration in the Parkinson’s model mice by suppressing microglial activation. Microglial activation is thought to mediate neurodegeneration by nitrated-α synuclein as dopaminergic neurons are degenerated. This is believed to lead to the inflammatory cytotoxic cascade and it seems as though the Tregs are able to interrupt this cascade and protect nearly all the remaining dopaminergic receptors. While reading this, I was curious if any research was going on to effect the differentiation of T cells into Tregs as a possible therapy for Parkinson’s. I found one article that shows a possible therapy in The Journal of Immunology by Magdalena Polanczyk, et al: Cutting Edge: Estrogen drives expansion of the CD4+CD25+ Regulatory T cell compartment. This study showed that by treating mice with 17- β-estradiol, their cells expressed more FoxP3 (both in vivo and in vitro), which in turn caused an increased expression of Tregs and FoxP3. The increased expression of FoxP3 is especially interesting in that it controls the development and function of Tregs which would help explain their increased expression as well.

In further researching the role of T cells in neurology, I found that T cells help maintain cognitive functioning in the brain. In mice that were deficient in T cells, their learning and memory was impaired. However, when these mice were treated with transferred T cells from wild-type mice, their learning and memory deficits improved. Furthermore, Lewitus, et al showed that in vitro neural tissue treated with Teffs inhibited long term potentiation in the hippocampus which could prove to be an effective therapy for patients with MS and Alzheimer’s.

1. “FoxP3.” http://en.wikipedia.org/wiki/FOXP3
2.Lewitus G., et al. CD4+CD25- effector T cells inhibit hippocampal long term potentiation in vitro. European Journal of Neuroscience. 26: 1399-1406 (2007).
3. Polanczyk M., et al. Cutting Edge: Estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment. The Journal of Immunology. 173: 2227-2230 (2004).
4. Reynolds, A. et al. Neuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson’s disease. Journal of Leukocyte Biology. 82: 1083-1094 (2007).

Alzheimer's Neuroinflammation and Neurovascular Damage Enhanced by Fibrin

The primary cause and clinical manifestation of Alzheimer's Disease (AD) is currently understood to be the beta-amyloid plaques that are so lethal to the nerve cells they surround. Additionally, a secondary event that may be just as detrimantal in the AD pathology is the tau protein neurofibrillatory tangles that are present in this disease. Now, looking at more inflammatory processes, specifically those linked to neurovascular damage, may allow even further understanding and treatment of AD by minimizing these acceleratory events.
In the paper "Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease", Paul et al show very thorough evidence that fibrin plays an active part in the disease and its progression. Something that is crucial to understand about this horrible disease that is demonstrated in this paper and really focused on in many others is that it is a "downward spiral" process where the events that are involved in the pathology are all rapidly increasing the disease as they enhance each other's potent effects. For example, it is understood that it is beneficial to the brain cells to use them and this promotes their own health; but, with AD, brain cells are degrading and therefore less used which only propogates their deterioration. Inflammatory events also often work this way, and certainly in AD this not only adds to the pathology, but maybe, better said, multiplies or exponentializes it. Fibrin, here, is shown to be quite active in this, especially as an indicator and even a cause of vascular damage in the brain, which can dramatically speed up the devastating destruction of the brain tissue, allowing more proteins and harmful products through the blood brain barrier and causing more cell loss. This paper was especially impressive because of the thorough degree to which it investigated its hypothesis. Namely, it not only looked at a mouse model with higher fibrinogen, the precursor to fibrin, but also one with affected plasminogen levels, which is the precursor to plasmin which promotes fibrinolysis, or the breakdown of fibrin. The model used both genetic and pharmacologic approaches to assay whether or not there was real substance to its hypothesis and went far beyond the mere few angles that could have been used to check this out.
I would also like to point out that this month's National Geographic (Nov 2007) has its cover story on "Memory - Why We Remember, Why We Forget" and really highlights some of the things that are crucial to look at when delving into anything related to memory and the brain. Plus, it has some cool perspectives from some very exceptional individuals on both ends of the spectrum related to this subject.

Multiple Sclerosis, Inflammation, Bee Venom Components

The article "The Relationship between inflammation and atrophy in clinically isolated syndromes suggestive of multiple sclerosis" caught my eye immediately, as this class has opened my eyes to how huge of an impact inflammation has on the functions of the body. In this case, the study investigated a possible relationship between inflammation (in patients with Clinically Isolated Syndrome) and brain atrophy; although the triple-dose gadolinium-enhanced MRI scan was found to be successful method, the hypothesis was found null: inflammation and brain atrophy do not proceed in parallel. This was surprising to me, I would think an inflammation disease affecting the brain and spinal cord would have an effect on the decrease in brain tissue volume, since it can have degenerative effects a lot of the time.
Inflammation, however, does have a serious effect on MS patients. Apitherapy has also sparked my interest into finding out why a bee sting could improve the quality of life of an MS patient. Bee venom contains extremely potent anti-inflammatory components, including melittin and adolapin. Melittin is a 100x more potent anti-inflammatory that hydrocortisone. It also has anti-microbial effects, including inhibiting the bacteria causing lyme disease, in which cytokine production is decreased and TNF-alpha is disregulated resulting in pro-inflammatory outcomes. Adolapin, also a highly potent anti-inflammatory, inhibits cyclooxigenases, which generate prostanoids and play a regulating role in inflammation. Apamin, another component of bee venom, is a neurotoxin that blocks Ca2+ activated K+ channels in the central nervouc system. Specifically, apamin blocks SK (small conducted) channels, which play roles in hyperrepolarization occuring immediately after an action potential. It seems to me that apamin in this case would be of more use to MS patients that the anti-inflammatory components of bee venom. Melittin and adolapin do not do anything specific enough to MS inflammation to be worth the therapy; however, the apamin's inhibitory effects dirrectly affect the CNS, which is what is directly affected by MS. If apamin inhibits the channels which play roles in hyperrepolarization, action potentials would then proceed at a slower pace, which could in turn slow down the degradation of the myelin sheath during MS. Therefore, I think bee venom seems to only be a decent therapy if used for slowing down the degradation process of MS, but as far as anti-inflammatory effects go, I have yet to be convinced it can be worth all those stings!

To Sting or not to Sting

In the Article titled A randomized crossover study of bee sting therapy for multiple sclerosis it was concluded that the bee sting therapy made no notable improvements on patients with relapsing MS. The study divided patients into two groups; for 24 weeks the first group was administered bee sting therapy and then for 24 weeks they were not stung. The second group received opposite treatment; first 24 weeks not stung, second 24 weeks received bee sting therapy. The stings were administered 3 times per week and each time the patients were stung once more than the previous time maximizing the amount of stings per session to 20 stings. After various methods of observation, it was concluded that bee sting therapy does not help and the article even says that patients with relapsing MS should refrain from this form of therapy until there is better evidence to support it.
Other articles found also suggest that bee therapy is a cloudy subject; whether or not it works is yet to be proven scientifically.

On the other hand, if you ask Pat "the bee lady" Wagner, bee sting therapy is a wonderful discovery. According to Pat, bee venom contains Melittin which "greatly improves vision, coordination, mobility, and sensitivity to touch, among other things, in MS patients. They also decrease pain, can add to a feeling of overall well-being, and even boost energy levels."

The general consensus on whether or not bee sting therapy works seems to be that it really depends on the patient. Some swear by it and others feel that the present evidence is just not adequate enough.

References:
http://health.discovery.com/centers/althealth/beetherapy/sclerosis.html

http://www.ahealthyme.com/topic/beevenom

Immune Tolerance During Pregnancy

Pathogens and foreign material are recognized by the immune system and can be rapidly cleared via innate/humoral and adaptive mechanisms. Yet during pregnancy, the embryo/fetus is protected and nurtured despite the expression of foreign paternal antigens. It is suggested that a state of tolerance without immune suppression is achieved between the mother and fetus/embryo, preventing both rejection and graft-versus-host-disease(GvHD).

Post fertilization, the embryo becomes surrounded by the zona pellucida which effectively creates an environment where substances can be excreted but the maternal immune system cannot access the expressed foreign paternal antigens. Tolerance of the embryo is not specific to the uterine cavity since implantation may occur in the fallopian tube, ovary or, rarely, the abdominal cavity. Even allogenic (as in donor ovum) embryos routinely survive to term in In-Vitro Fertilization procedures despite containing entirely foreign antigens.

The recent literature indicates that pregnancy does not generate immune-suppression but rather a state of controlled inflammation. During implantation, the endometrium is remodeled to allow blood vessel penetration. The remodeling can be so aggressive that maternal immune response (Th-1) is needed to prevent over-invasion. However, if the immune response shifts to Th-2, over-suppression occurs resulting in decreased angiogenesis and embryo rejection.

This shift is an important mechanism for the expulsion of defective or seriously infected embryos. It is estimated that more than 95% of defective embryos are terminated early due to the lack of proper immune tolerance. Barnea shows that an embryo-derived protein, preimplantation factor (PIF), has a role in initiation of maternal tolerance and receptivity of the uterus to implantation. This is the first time that a clearly embryo-derived factor has been found in early pregnancy (in the embryo/fetus and placenta) and also in maternal peripheral circulation. Presence of PIF in maternal blood within 4 days of embryo transfer, increased the delivery rate to more than 70% to term versus the 3% delivery rate if implantation was delayed.

It has been shown that symptoms of some autoimmune disorders improve during pregnancy. Women with Multiple Sclerosis (MS) show major symptom improvement in the third trimester but experience rapid flare-up after delivery. Insulin requirements for women with juvenile diabetes mellitus (JDM) decrease 12% during early pregnancy but then increase to 50%. Interestingly, PIF also has the ability to modulate the immune response in autoimmune disorders and prevents the development of GvHD in semiallogenic animal transplants.

In the mouse autoimmune encephalitis (EAE) model for MS, low-dose short-term PIF administration on non-pregnant mice reduced paralysis, delayed initiation of the disease and increased the survival of the animals. IL-12 is a major inflammatory cytokine implicated in MS, and PIF also decreased the IL-12 serum level. PIF’s mechanism in JDM acts to protect insulin producing Langerhans cells from destruction. PIF therapy also prevented skin ulceration, hepatitis and severe weight loss in a GvHD model that transferred semiallogenic immune cells to irradiated mice.

This 15-amino acid peptide appears to modulate immune tolerance in organ systems as varied as spinal cord, pancreas, skin, liver and bone marrow. It was shown to be non-toxic, even at very high doses in vivo, and can be easily synthesized. Which suggests it could be a prime candidate for drug development in the treatment of autoimmune disorders. The research in this area is ongoing (manuscripts in preparation) and may transition from preclinical to clinical trials in the near future.

Barnea, E.R. Applying Embryo-Derived Immune Tolerance to the Treatment of Immune Disorders. Ann.N.Y.Acad.Sci. 1110: 602-618(2007).

Influence of age and physical activity on the primary in vivo antibody and T cell-mediated responses in men.

In older human populations, both the immunoglobulin (Ig) response and delayed-type hypersensitivity (DTH) to antigenic challenge are altered. As a result, older persons are often inadequately protected from diseases because of ineffective responses to vaccination.

As you may recall, when antigen is presented to the body, the B cell first produces IgM. The B cell then undergoes antibody class switching via T cell help in order to produce IgG. In humans, there are 4 subclasses of IgG, and each differs in its function and cytokine requirement. If IgM production improves with physical activity, it’s possible that there’s improvement in antigen-presenting cell and B cell interaction. On the other hand, if there’s a change in an IgG subclass response, then it might indicate changes in Th1 or Th2 cytokine production.

Through the use of keyhole limpet hemocyanin (KLH), a benign T cell-dependent protein, and 46 active and sedentary, young and older men, this particular study consisted of 2 “sub” studies that (1) sought to verify that KLH immunization in humans would stimulate detectable and antigen-specific antibody and DTH responses, and (2), tested whether there was an age-associated decline in the primary antibody to a novel antigen; an age-associated decline in memory T cell-mediated responses to a skin challenge (DTH) with the antigen; an effect of regular physical activity on these antigen-specific responses; an association of age and physical activity in antigen-specific antibody isotype (IgG and IgM) and subclass (IgG1 and IgG2); and differences in specific antibody response that can be accounted for by total circulating IgM, IgG (1, 2, 3, & 4) in aging and physical activity.

The take-home message from this study, overall, is that physical activity in aging human males is associated with elevated generation of a primary antigen-specific T cell-dependent antibody and DTH responses. In more detail, and taken directly from the literature, the authors observed that there was an age-related decline in the primary antibody response to the novel antigen, KLH; that there was an age-related decline in the memory T cell response to KLH; that older physically active subjects have an improved antibody and DTH response compared with older sedentary subjects that is equal to younger subjects; the changes in anti-KLH IgG production are primarily of the IgG1 subclass; and that the detected changes in antibody are reflective of improvements in antigen-driven responses because total antigen nonspecific Ig was not affected.

Thus, regular physical activity in older males is associated with a stronger immune response to novel antigenic challenge.

Smith, Taro P., Sarah L. Kennedy, and Monika Flshner. Influence of age and physical activity on the primary in vivo antibody and T cell-mediated responses in men. J Appl Physiol 97: 491-498, 2004.