Microcluster Condensation in Immunological Synapses 1

I remember reading one of the popular science magazines I subscribed to back in high school, probably either Popular Science or Discovery, and coming across a brief article on the discovery of the immunological synapse. It included pretty pictures. I was intrigued by the spatial and sequential alignment of disparate signaling effectors, even though I knew absolutely nothing about the context at the time. Now, I know more about the molecules and pathways involved in dendritic cell::T-cell signaling than I am able to concisely put down here in words without rolling myself up in a cloak of jargon, and even so I barely know anything (with comparison to both the experts in the field and the scale of the unresolved questions). The immunological synapse is fascinating, and to me it is beautiful in its absolute parsimony (that's a whole other post for later).

In beginning the activation of the adaptive immune system, dendritic cells process and present sampled antigen in distinct molecules (MHC) that T-cells can recognize (via the TCR and CD4/8). Due to chunk recombination of V, D, and J regions of the TCR binding motifs and subsequent pre-programmed random mutagenesis* there is extremely high heterogeneity in the recognition cognates of the TCRs. So as dendritic cells (DCs) crawl through the thymus, lymph node, spleen, or other, they have many distinct antigens loaded into their surface display molecules, and every once in a while a TCR that has some binding affinity for that antigen will bind. What follows is the immunological synapse.

The immunological synapse starts out with the binding of the TCR and CD4/8 to the MHC, which nucleate the formation of the central supramolecular activation complex (cSMAC), when all of the TCR/MHC complexes from microclusters and merge into 1 more stable site. A lot of other things happen downstream of that, most of which are very interesting**, but what I find intriguing about this is: what sort of topology do the kinetics of microcluster condensation add up to?

All optimized networks have some sort of topology. This means the hierarchy of one node over another, because to have all nodes processing the same exact bandwidth is rather energetically inefficient. As such, there can be strictly hierarchical topologies like those found inside human corporations with management, there can be scale-free topologies in which hierarchy arises due to through-put optimization and is not strict (a good example of this is the server structure of the Internet), or others I don't know anything about yet.

Is microcluster condensation hierarchical or scale-free?

Unfortunately, it is extremely difficult to answer that question empirically because it happens so fast and because the cells involved are rather camera-shy unless given very exacting and munificent conditions. Therefore, this is more or less a thought experiment.

I posit that microcluster condensation is both hierarchical and scale-free, in turn; first, hierarchical and then, later, scale-free. Cook your noodle on that for a bit, and I'll explain my reasoning for why within the next few days.

*Not a contradiction of terms. The immune system allows for random mutagenesis of a restricted set of amino acids residues on T-cell receptors and B-cell receptors to greatly increase the range of possible binding motifs without great additional informational storage costs (DNA).
**E.g., I find the activation of such factors as NFAT, mTOR, et al to be interesting, but generally find the dynamics of histone deacetlyation to be rather dull.

Your Microbiome and You

You are never alone. Not even when you might want to be. Tucked away within the ~100m² of your bowels are ~10¹⁴ (there are ~10¹³ somatic and germinal cells in the human body) of your closest friends, collectively termed The Microbiota. They eat, spawn, conjugate, die, poop, fight, and secrete right there inside of you, unseen and mostly unthought of except when something is wrong. This system, the remarkably homeostatic mammalian gut, forms what is perhaps the densest and most complex microbial ecology on this planet.

These teeming microbes are not mere freeloaders living off of your access at their own convenience, they are true symbionts. In exchange for a warm, wet home and nutritional supply, they break down starches for us, metabolize complex molecules, and synthesize some key compounds, such as Vitamin K. It has been found that gnotobiotic, or germ-free, animal models require ~30% more calories to develop normally without a microbiota to help them out. In humans that have been on a broad-spectrum antibiotics, hardier inhabitants (such as Clostridium difficile) can bloom when all of their more sensitive neighbors (such as Bacteroides spp. and Bifidobacterium spp.) are killed off, which causes very unpleasant colitis and diarrhea, that can then be cured by a transplant of fresh microbiota from a healthy individual (colloquially referred to as "poop soup"). Microbiome transplants can also transfer physiological characteristics from one individual to another. For example, the microbiomes of obese individuals have been found to have reduced numbers of Bacteroidales spp., and transfer of these microbiota via poop soup into germ-free mice resulted in obese mice, theoretically because these microbiota were more efficient at releasing calories from food.

Microbes exist, or can exist, in virtually every segment of the gastrointestinal tract from mouth to anus. In the mouth, a variety of Actinomyces spp. are associated with the formation of plaque. In the forbidding and harsh environment of the stomach, only Helicobacter pylori can thrive (it does so by hiding among the mucous lining the stomach and modulating the host immune response) and it has been found to directly cause stomach ulcers and has been further implicated in the formation of gastric cancers (it's the only organism classified as a BSL 2+ carcinogen). The proximal portion of the small bowel is relatively sparsely colonized at ~10⁴-10⁵ microorganisms/ml lumenal contents, which contrasts sharply with the densely colonized colon (~10¹⁰-10¹² microbes/ml contents).

In the human and other mammals, diverse and distinct microbial ecologies also exist in the sinuses, ears, genitourinary tract (largely Lactobacillus spp. in the vagina; the bladder is generally only colonized in disease states [long-term catherization and/or pyelonephritis] by uropathogenic Escherichia coli, Proteus mirabalis, et al), and on the skin as a whole (mostly Staphylococcus spp.). These others will, however, be excluded from the present discussion.

However, what's very puzzling about all of this is: how does the mammalian immune system manage to differentiate from the massive basal antigenic signals coming from the microbiome from pathogenic antigens? In other words, why isn't the immune system raging against the huge number of microbial signals in the gut?

One of the exquisitely elegant features of normal gut physiology is that gut-associated lymphatic tissues (GALTs) mediate fine-tuned hyporesponsiveness to commensal microbiota while remaining responsive to pathogenic microbes. This flies directly in the face of most immunology, which holds that microbial antigens will always provoke a stimulatory response when ligated to TLRs, CLRs, or NODs (conserved receptors of the immune system that bind conserved molecular patterns associated with pathogens). In vitro data support this. Physiology doesn't.

Physiologically, the germ-free mouse is weird. A germ-free animal is one that has been reared in an environment completely free of all microbes, fungi, and exogenous viruses and as such they have no native intestinal microbiota. Not only do they require more calories and vitamin supplementation, but they also tend to accumulate undigested fibrotic material in their ceca, which predisposes them to gut twists and bloat. Additionally, they feature underdeveloped Peyer's patches (distinct GALT sites on the gastric mucosa), altered CD4+ T-cell and IgA-producing B-cell population profiles, and the follicles in the spleen and lymph nodes where T- and B-cells mature are poorly formed. All of these abnormalities can be rescued by adding back microbial signals such as LPS, even without the microbes themselves. Due to these alterations, it is becoming accepted that the microbiome plays a crucial role in the normal development of the immune system. But to reconcile this with the dogma of microbial signal + PRR ---> inflammatory immune reaction is somewhat difficult, or at the very least complex.

Immune cells that reside in the lamina propria underneath the gastric epithelium generally show signs of recent activation and a particular subset of dendritic cells (CX3CR1+) has been found to extend dendritic processes up through the tight junctions binding gastric columnar epithelial cells together to directly sample the lumenal contents. M cells that cap the Peyer's patches have been found to shuttle lumenal contents, and any antigens contained therein, to the dendritic cells and lymphocytes underneath. These pathways of antigen exposure are thought to be involved in the induction of immunological tolerance to microbiotal antigens, which could explain why the immune system does not attack the commensal microbiota. However, it does not explain how pathogen antigens processed by the same pathways are recognized as pathogenic and stimulate the immune system to attack.

Recent evidence strongly suggests that the intestinal epithelium itself is responsible for the differentiation of nonpathogenic microbiota from pathogens. Canonically, the intestinal epithelium is thought of as a simple barrier that is involved in the absorption and transcytosis of metabolites and nutrients. But it seems that it is much more involved that we had previously believed.

It turns out that intestinal epithelial cells (IECs) express TLRs and directly modulate the composition of the microbiome itself as well as the responsiveness of immune cells. This ranges from TLR expression on Paneth cells in the small intestine that secrete potent antimicrobial molecules (RegIIIg) when ligated [Dr. Lora Hooper, in seminar given 11/19/08] to actual expression of MHCII and direct antigen presentation. It was previously believed that MHCII expression was restricted to antigen presentation by dendritic cells.

When investigators deleted TLR4, NOD1, or MyD88 (an adapter protein involved in many TLR-mediated NF-kB inflammatory pathways) in murine IECs they found that the mice were more susceptible to bacterial infections, which implies that the TLR signalling on the IECs is essentially to the development of normal protective immunity. A second feature of this is that IEC TLRs and NODs are located intracellularly, instead of on the cell surface as in immune cells, which means that they'd only be ligated and activated when an invasive pathogenic microbe breaks into the IECs themselves (e.g., Salmonella typhimurium, Vibrio cholerae) as opposed to the more peaceful commensals. It may be that noninvasive gastrointestinal pathogens are recognized by the proteins that they shoot into IECs via Type IV secretions systems (e.g., Tir and Escherichia coli O157:H7) in the same manner.

The commensal microbiota is also at work on the IECs themselves, actively acting against IEC-mediated inflammation. Bacteroides thetaiotaomicron has been found to induce the PPARg anti-inflammatory (acts by increasing cytoplasmic shuttling of pro-inflammatory NF-kB away from the nucleus) mechanism in vitro. Commensal-derived metabolites such as butyrate (a short-chain fatty acid) have been found to inhibit expression of pro-inflammatory cytokines and increase expression of anti-inflammatory cytokines in IECs.

It is now thought that IECs regulate dendritic cell function through secretion of thymic stromal lymphopoietin (TSLP) and modulate T-cell activity through expression of MHCII in the abscence of costimulatory molecules. TLSP acts directly on dendritic cells and inhibits their production of pro-inflammatory cytokines (such as IL-12), which in turn promotes dendritic-cell-mediated activation of regulatory T-cells. TSLP is also implicated in skewing the immune response to a T_H2-type T-cell response, which is implicated in both response to metazoan parasites and pulmonary atopy. If naive T-cells are being exposed to MHCII on IECs without co-stimulatory molecules, then the T-cells will either kill themselves off (anergy) or mature into tolerogenic T-cells that limit the immune response to those given antigens. This, combined with widespread TGFb secretion by IECs, directly indicates an active role for IECs in promoting immune system hyporesponsiveness to the antigens present in the gastrointestinal system. Without this direct suppression of active, inflammatory immune responses, the immune system would be in a continual inflammation state due to not knowing what to do with a safe commensal antigen vs. a dangerous pathogenic antigen. Indeed, emerging research indicates that dysregulation of this process may underlie the pathophysiologies of inflammatory bowel disease and Crohn's disease.

It'll be interesting to see what's found next.

Artis, D. (2008). Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut Nature Reviews Immunology, 8 (6), 411-420 DOI: 10.1038/nri2316

How Vaccines Work: An Immunological Primer

Vaccines prevent disease. They do so by causing you to develop immunity against an infectious organism before it gets a chance to infect you and cause disease. In essence, vaccines teach your immune system how to recognize infectious organisms and fight them off much faster. Without vaccination, exposure to an infectious organism is like being thrown into the deep end of a pool without water wings where your immune system thrashes around desperately trying to save you. But vaccines are like the shallow kiddie pool where you touch the bottom and your immune system gets acquainted with the water so that it will have a better idea how to handle it later on when you can't touch the bottom. Vaccines do this by exposing the immune system to dead, weakened, or pieces of infectious organisms, usually along with a signal molecule called an adjuvant, so that it can learn that the molecules associated with the infectious organism are bad and should be attacked if they're ever seen again. The adjuvant acts like a wake-up call to the immune system to make sure it takes a look and does what it's supposed to.

Vaccines are education for the immune system.

The human body has 2 different immune systems: the innate immune system and the adaptive immune system, and they talk to each other quite a bit. The innate immune system is the infantry of the immune system; it reacts rapidly to invading pathogens and can destroy most of them, but it can have a hard time telling exactly who to kill and when and how much firepower to use. Meanwhile, the adaptive immune system is more like a laser-guided cruise missile from a fighter jet that very specifically takes out invading pathogens and tags the enemies that the innate immune system should kill off. However, just like it takes time for jets to get to where they need to go, the adaptive immune system takes several days to warm up and start fighting.

The innate immune system is scattered throughout the body in circulating white blood cells, in the mucous membranes, in the skin, and in the gut; all places where invading pathogens are likely to try to enter. The adaptive immune system, when there's no infection present, generally hangs out in the bone marrow, thymus, and lymph nodes; but when there is an infection some of them will go out to the site of infection to join the battle while others will stay behind and direct subsequent reinforcements.

In an infection where there is no prior vaccination present, say S. aureus in a pimple, the innate immune system quickly (in minutes) recognizes many of the molecular patterns that are generally associated with all bacteria, including S. aureus. These patterns are signals for the innate immune system to kill and also go talk to the adaptive immune system. So the innate system goes to work blasting away as well as it can at the S. aureus, but it's good at hiding so the innate system doesn't see all of it and S. aureus is able to establish an infection.

So meanwhile, the innate immune system has gone off to the adaptive immune system and handed it the information it has gathered about the infection. The adaptive system examines it and then begins to multiply and specialize. Some of the multiplying cells get out to the site of infection and join the battle. Other cells make antibody against the S. aureus, which is then secreted into the blood and binds the bacteria, lighting them up brightly and saying "Destroy this now!" to the innate and adaptive system cells that are already there.

Once the infection is cleared, many cells of the adaptive immune system specialize again and become memory cells that wait out in the body, able to specifically recognize the same infection they just fought off. This means that if S. aureus tries to come back, the memory cells will see it very specifically and very quickly and they can then jump-start the rest of the adaptive immune system and mount a very rapid lethal attack against it.

The problem is that many infections are strong enough to kill, or do serious and permanent damage to, a human before the adaptive immune system is strong enough to fight it off. Although the innate immune system starts fighting quickly, it takes the adaptive immune system about 4 days to start responding very much and a full 7 days to really get into swing. Memory immune cells can bypass that waiting period and kick both immune systems into battle mode right away.

Vaccines are designed to skip the infection part and go directly to teaching the adaptive immune system to recognize the infectious organism and then form the memory immune cells that will kill the pathogen before it even has a chance to cause infection.

This is why vaccines are effective, useful, and necessary. They also happen to be quite safe*.



NOTE: This was largely inspired by Abel Pharmboy's guest post over at Dr. Isis' place. I left a long comment there and then realized that it wouldn't do very well for me to be a hypocrite. If you feel I've left anything out, or if there's anything you want to know more about please let me know. I tried to keep it simple because even the basics of how the immune system functions could keep me busy with posts for the rest of the year.

*Bears, however, are something you should worry about. Bears are crazy dangerous.

Allergy Inaccuracy (Toaster Burns Bullshit #3)

I need to stop reading anything that appears on the Huffington Post or else I fear the vein running through my right temple will cease merely throbbing and commence with exploding all messy-like everywhere. I sent them some email expressing my anger and disgust over the absolutely abysmal science/medicine reporting standards they apparently have, as have many others in the sciency blogosphere. Apparently they're being reactionary, recidivist bastards because the shit just keeps getting deeper and worse and more stupid that I had believed possible. It is as though Toaster's Stupidity Scale, tempered by the rhetorical vomit and denialist vehemence of the last 8 years, has had to redefine a new Absolute Zero for this kind of stuff.

Not only has the Huffington Post run a poorly informed and incredibly biased article by Jim Carrey (much better known as The Mask and Ace Ventura than a medical expert) on the autism-vaccine link, they're also pimping a link to some site called Intent with an article entitled "The Self Against the Self: Deal With Seasonal Allergies Naturally and Cheaply". The Sciborgs Orac, Abel Pharmboy, and PalMD have ably knocked the wind out of the "ZOMGVAXXIENZ!" argument cloud fogging Mr. Carrey's brain. So I will be burning the latter article.

"The Self Against the Self: Deal With Seasonal Allergies Naturally and Cheaply" contains some vague mystical advice about how to use Zen and Detoxification to get rid of allergy symptoms. It was written by one Debbie Mandel (website), author of several spiritual self-help books, and whose Intent bio says she will help you build immunity to feeling bad. Note that she is not an immunologist, nor even a biologist, but she'll tell you how to build immunity anyway. I wouldn't have a problem with this claim because building immunity can, sometimes, have a different connotation of inuring one against something unfavorable IF she hadn't actually tried, and failed epically, to invoke actual immunology.

Your response to allergens correlates to how balanced you are regarding: eating, exercising, sleeping and stress-management. Allergies are actually an auto-immune response which translates into: the self against the self. [boldface hers]

First the less egregious problem about homeostatic health and allergies: No, allergen responses don't much depend on whether you're stressed out about work or not. Just ask someone with an acute peanut allergy. Allergic responses are, however, modulated through general systemic inflammation if it exists, but this isn't much relevant unless you're morbidly obese (excess fat can increase inflammation).

This second claim, though, is either sheer stupidity or inability to do research. Allergies and autoimmune disorders such as rheumatoid arthritis are NOT the same thing. Some cases of autoimmunity may have similar underlying immune responses as allergy, but this does not mean they are alike. It is true that allergies are an abnormal immune response, but not against the self. Allergens are strictly defined as harmless environmental substances that evoke an immune response as if they were harmful. Or, as a professor once put it, allergens are nonharmful nonself being treated by the immune system as though they are harmful nonself.

The human body, and its immune cells, come into daily contact with millions of nonself molecules and doesn't have an allergic reaction to them. The immune system is generally quite good at distinguishing between what can harm us and what cannot. Allergies arise when the immune system mistakes something that is harmless, like pollen, as harmful and reacts to it.

Here's how allergies basically work (my explanation):

1) Dendritic cells present antigen in MHCII to CD4+ T-cells all the time without much caring what they're presenting. Sometimes harmless antigen gets presented.
2) Some naive CD4+ T-cells are, by virtue of the recombination and mutagenesis that occur as the T-cell receptors are being formed, naturally reactive against that harmless antigen.
3) Harmless antigen-reactive CD4+ T-cells proliferate and usually differentiate into a TH2-type response. The TH2 T-cell phenotype is characterized by secretion of IL-4.
4) TH2 T-cells talk to B-cells and get them to make antibodies against the harmless antigen, now an allergen. Allergen-specific antibodies are usually of the IgE isotype.
5) IgE gets made and secreted into the plasma, where it can become the receptor for mast cells and basophils. Eosinophils get involved in this, too.
6) When an allergen binds mast cell-IgE, it signals the mast cell to release allergy symptom-causing histamines, prostaglandins, and leukotrienes. These chemicals are generally what allergy medications try to work against.
7) Some allergen-reactive TH2 T-cells and their corresponding B-cells will become memory cells, ready to spring back into action at the drop of their allergen.

At no point in this is the immune system reacting against host antigens. In fact, the immune system tries very hard to kill all host-reactive T-cells as they're being made (this results in >99% of all developing T-cells being killed off). If Debbie Mandel had done her research before making up the above assertion, she'd have known better. This is irresponsible, dishonest, and lazy. Although I guess the immune system reacting against harmless environmental substances just isn't as fear-inducing as a self-reactive immune system.

Your response to allergens correlates to how balanced you are regarding: eating, exercising, sleeping and stress-management. Allergies are actually an auto-immune response which translates into: the self against the self. So, it makes absolute sense that when you are intact and whole, your allergies will dissipate and no longer bother you; your immune system will be more vigilant. I present myself as a test case.

It only makes sense if you're poorly informed. Memory immune cells last for many many many years, or else vaccines wouldn't work at all. Presenting herself as a test case is also stupid. How many times do we need to tell people that anecdotes do not equal evidence!?

For years I was an allergy sufferer. My sinus headaches, dizziness, itchy eyes and occasional tiredness always arrived with spring. Every morning I woke up to coffee and Allegra D 180 mg. However, in the past few years I rarely have any symptoms except for the occasional itchy eye and more important, no more killer headaches and sinus pressure!

What has changed? I stopped taking the medication five years ago, ate more fruits and vegetables and basically stopped fighting Mother Nature; I vowed to get along with her no matter what the weather. Brazenly, I went out to my garden, in the trenches, fertilizing, weeding, mulching and pruning without the fear or the expectation of an allergic reaction. I made up my mind not to have any symptoms and oh yes, I almost forgot, I managed my stress.

Note that she doesn't mention how long it took her to stop having symptoms. In all probability, she was quite miserable for the first couple years. What she did here was not Mystical Woo Magic, it was a clumsy and messy version of hyposensitization treatment. In clinical hyposensitization treatment, patients with allergies are inoculated with progressively greater doses of purified allergens. Hyposensitization does not exactly cure allergies, but it does relieve symptoms by skewing the immune response away from a TH2 IgE to a TH1 IgG, which doesn't rely on the mast cells or basophils that can mediate allergic rhinoconjunctivitis. Ms. Mandel would have gotten the same results clinically (but that would have meant vaccine-like injections, which might contain Teh Mercury and Teh Aluminumz! ZOMGs!).

Ultimately, this might have been an interesting and useful article if Debbie Mandel had done their research properly. As it is, her failure to do so has made her writing as flimsy as her detoxicification and Teh Evil Sugarz screeds.

Post Script: Please note that I do not have a problem with people writing about the immune system in the popular media. In fact, I support the information and principles getting more exposure because I think the immune system is The Awesome Eleventy Coolness. If I could, I'd pass out the DC-->T-cell-->B-cell-->antibody dogma in tasty ladlefuls on the street corners if it could be made into a pudding. However, I have a big problem with non-scientists who are completely unqualified trying to assume professional authority and opine about the mechanics of the immune system, or any science for that matter, when they have not done the research thoroughly. That's just irresponsible.

Dendritic Cell Phagocytosis of Infected Apoptotic Cells Favors Development of a TH17 Phenotype

Background: TH17 discussed previously here; previous brief run-down of T-cell development here.

TH17 cells are an inflammatory T-cell subtype implicated in acute adaptive immune response as well as chronic autoimmune diseases. We have known for a while now how to create TH17 cells in a dish: just add TGFb and IL-6 (a general T-cell proliferation cytokine such as IL-2 wouldn't hurt either). However, we haven't been very clear on how exactly those kind of conditions would arise in vivo. TGFb is anti-inflammatory and helps slow the adaptive immune response down while IL-6 is pro-inflammatory and revs up the immune system. So how would these 2 molecules be made at the same time? Logically speaking, they wouldn't as it is tempting to think of the immune system as monolithic: capable of one state at a time, either ramping up inflammation or cooling it down. But because TH17 cells have been found in vivo infiltrating tissues and being made in the lymph nodes, and because we haven't yet found any other combination of cytokines that leads to the thorough development of TH17, TGFb and IL-6 must be co-expressed under some condition, somehow.
____________________________________________________________________
Before we get there, I'm going to blather at ya'll about dendritic cells for a moment (although I wish I BlogSpot had the functionality to embed this in a text-wrapping box). Dendritic cells (DCs) are the interface between the innate and adaptive immune systems. DCs are phagocytic, meaning they can gobble up pretty much any cell that they see fit to do so to. When they gobble something up, they put it into a special vacuole and crank it through molecular blenders (called proteasomes) to generate short little peptides that they then port back out to their surfaces on major histocompatibility II proteins (MHCII) that T-cells can bind to and recognize. T-cells can't recognize peptides, called antigens here, without the context of MHCII. Correct peptide-MHCII-CD4-TCR signalling tells the T-cell to do adaptive immune stuff that helps the body fight off disease.

Figure A: A dendritic cell grown in vitro on collagen. Note the tentacley things protruding all over the place, those are the dendritic processes. Not all of the dendritic processes in this picture are from that one cell as other dendritic cell bodies are off screen. Picture somewhat altered for better contrast from original Wikimedia Commons jpeg.

DCs also possess Toll-like receptors that recognize PAMPs (pathogen-associated molecular patterns). PAMPs include dsRNA (TLR3), flagellin (TLR5), single-stranded RNA (TLR7), unmethylated CpG DNA (TLR9) and, most importantly here, LPS (TLR4) [LPS is a constitutive component of Gram-negative bacteria, which shed it. It is also known as endotoxin.]. When a TLR binds its ligand, the DC gets activated and migrates to the lymph node where the naive T-cells are hanging out. Once there it secretes various effector cytokines that have specific actions on the T-cells. These cytokines include IL-2, IL-4, IL-6, IL-12, TGFb, TNFa, and a whole other mess of alphanumeric soup noodles.
____________________________________________________________________

Anyway, back to the conundrum of how inflammatory IL-6 and anti-inflammatory TGFb get co-expressed and make TH17 cells in vivo. It turns out that infected apoptotic cells can lead to the right co-expression. DC processing of apoptotic cells induces TGFb to throttle down inflammation in case the immune system has gotten too aggressive and is killing off the host organism (as occurs in septic shock). DC processing of non-host infectious agents usually induces IL-6. So when a DC processes an apoptotic cell that was infected with an intracellular microbe of some sort, both stimuli are right there and the DC trundles off to the lymph node making both TGFb and IL-6.

The group in the paper below used LPS-loaded apoptotic B-cells and E. coli-infected apoptotic neutrophils to test this out. They mixed the B-cells and neutrophils with DCs and let them stew for a while as the DC chewed thoughtfully and decided what to do. They then collected the culture medium off fo the DCs, which would contain any differentiation and/or signalling cytokines the DCs secreted in response to the apoptotic cell stimulus. Next, this medium was added to cultures of naive CD4+ T-cells and they sat back and watched what happened via flow cytometry, mRNA expression, and production of phenotypic marker cytokines (specifically, IL-10 and IL-17).

Using unloaded apoptotic B-cells or uninfected apoptotic neutrophils didn't do much by way of making TH17 cells as only TGFb was produced (made Tregs, though, see below). However, this was reversible through complementation with exogenous IL-6. Also, DCs exposed just to LPS such that TLR4 was strongly activated did induce lots of IL-6 production, but this didn't lead to much TH17 production unless IFNg was blocked (this consequently blocks the production of TH1 cells).

It turns out that apoptotic cells also induce a Treg response through TGFb. Tregs are the cells that calm the rest of the immune system down when it's gotten too excited. They are characterized by expression of Fox3p and secretion of IL-10. A sizeable portion of the naive T-cells also made IL-10 and flow cytometric analysis revealed that some had become double T-cells, secreting both IL-10 and IL-17. However, this was only during the initial stimulation of the naive T-cells. Later on, after these now-differentiated T-cells had calmed down, re-stimulation with IL-23 increased IL-17 production and downregulated IL-10 mRNA, indicating that this dual expansion may be a transient phenomenon.

The group replicated their findings in vivo. They used a Citrobacter rodentium model of hemorrhagic colitis in which many cells in the gastrointestinal epithelium go apoptotic and inhibited that apoptosis. Inhibited apoptosis led to lower infiltration of TH17 cells than untreated mice. There were also a bunch of good genetic and chemical controls, but I'm not going to discuss all of that here because 1) it's a LOT of detail and 2) I'm more interested in blathering about the implications of this finding.

Implication 1: Ulcerative colitis
The sick gut cells in ulcerative colitis aren't necessarily infected themselves. But due to the very high native colonization of the gastrointestinal tract by our friendly microbiota, there are always relatively high levels of LPS in the lumen that, presumably, interact with the epithelium. Therefore, at the ulcers, especially those mediated by Helicobacter spp., there is an increased likelihood that responding DCs will encounter both microbial PAMPs and apoptotic cells. This could explain, at least partly, why TH17 cells are heavily involved in the inflammation associated with gastritis.

Implication 2: Cancer
Given that pre-cancerous cells are generally not listening to the cells around them and have higher rates of mutation, it is entirely plausable that some cancer cells with alter the expression patterns of MHCI on their surface, be recognized by CD8+ T-cells, and summarily executed. However, were a DC to encounter this apoptotic body in the abscence of a TLR ligand, it would go to the lymph node promoting development of Tregs for the cancer cell antigens*. While this may be a good thing in preventing autoimmune reactions when T-cells learn the unmutated antigens still present in cancer cells are bad and should be killed, it also brings up the somewhat unsettling prospect that the this leads to immunotolerance of cancerous cells. The immune system is generally the first line of defense against the development of tumors, and if it has specifically learned to not be reactive against them due to Tregs, then the cancer would be allowed to continue growing and mutating without immune system interference. Needless to say, this is ultimately bad for the organism.

Implication 3: Rheumatoid Arthritis
It is known that inflammed tissues produce IL-23, which encourages stable differentiation of TH17 cells and also acts as a chemoattractant for them. I don't think it's unreasonable to think that levels of IL-23, along with NFkB et al, would be elevated in arthritic tissues and that this could lead to infiltration of TH17 cells. Once there, TH17 cells can cause further inflammation, including tissue damage. This in turn would amplify the production of IL-23, which has been shown to decrease IL-10 production and in turn decrease Tregs. This then leads to a dismal scenario in which more and more TH17 cells are being recruited to drive inflammation in arthritic tissue while at the same time stomping on the Tregs that could help break that feedback loop. However, at the same time DCs would be present and processing the apoptotic cells in the arthritic tissues, and then hopefully driving towards TGFb and Tregs.

It should be noted that each and every implication I have put up above could be complete and utter horsefeathers as the immune system is so complex and operates on so many scales that I could very easily be overlooking a key component that makes all of my conjectures seem silly.


Torchinsky, M., Garaude, J., Martin, A., & Blander, J. (2009). Innate immune recognition of infected apoptotic cells directs TH17 cell differentiation Nature, 458 (7234), 78-82 DOI: 10.1038/nature07781

*This happens anyway, MHCI or not. B7.1 and B7.2 (CD80 and CD86, respectively) expression patterns are often altered in chronic myelogenous leukemia and chorionic gonadotropic, which isn't expressed in adults, sometimes pops back up in tumors.

IL-23 --> TH17 +++ IL-23 --> TH1

By now it has become very well established that naive T-cells can and do differentiate into many many many different terminal states, although most of them will die along the way. A naive T-cell arriving in the thymus expressed both CD4 and CD8, and has to choose based on intercellular interactions with APCs (antigen-presenting cells) and the cytokine environment whether to be CD4+ or CD8+ and wind up recognizing antigen on MHCII or MHCI, respectively. CD4+ T-cells can then further differentiate, based again on cytokines, whether to become TH1, TH2, Tregs, or Th17 cells, or some memory variant of the first 3 types. TH1, TH2, and TH17 are all inflammatory phenotypes; Tregs are anti-inflammatory in that they restrain the adaptive immune response from going beserk and killing off the rest of the organism (e.g., septic shock). There're several cans, maybe even a tanker truck, of alphanumeric soup for all the various cytokines, CDs (cellular domains), transcription factors, etc. that regulate T-cell differentiaton. But today we're going to talk mostly (because there'll probably be others that necessarily slip in) about TGFb (transforming growth factor beta), IL-6 (interleukin-6), IL-12, IL-17, IL-23, and its receptor: IL-23R. It should be noted that IL-23R is a homodimer of the IL-12Rp40 (one of 2 subunits of the Il-12R) and a unique IL-23 heterodimerizing domain (p19).

So if proliferating, un-fully-differentiated T-cells are exposed to a mileau of TGFb, IL-6, and IL-21, they generally start to look more like TH17 cells based on their surface molecule expression and cytokine secretion. TH17 cells become TH17 cells when they start to secrete IL-17 (oddly, though, neither TH1 nor TH2 cells are characterized by their secretion of IL-1 [which is a generally pro-inflammatory cytokine] or IL-2 [which is a paracrine {usually} T-cell pro-proliferative signaling cytokine]). But TH17 cells aren't very good at being TH17 cells unless they can detect IL-23.

The group discussed in the paper generated IL-23R knockout mice and tested their TH17-like cells for various parameters. It turns out that without IL-23R (effectively the same thing as having absolutely no IL-23 as far as we know), TH17-like cells don't proliferate very well in the lymph nodes where they'd normally do so. This failure to proliferate as massively as they otherwise would means that there are fewer TH17 cells in circulation and thus fewer are available to infiltrate target inflammed tissue. However, no IL-23 signaling apparently doesn't affect the molecular markers that effect tissue migration, so it seems that IL-23 is primarily involved in driving lymph node expansion of activated TH17 cells. Without IL-23, TH17 cells are also much weaker in that they go apoptotic much more readily. Oddly, the group also reported that in vivo activation without IL-23 didn't affect Foxp3 expression, which I don't understand the relevance of because Foxp3 is primarily a marker for the development of Tregs.

Backing out to a broader picture: it has been found that inflammed tissues have high IL-23 expression. It has also been experimentally determined that injected IL-23 into the skin results in IL-22 mediated dermal inflammation. TH17 cells produce IL-22. So it looks like IL-23 in inflammed tissues may be attracting TH17 cells to come and check it out, then expand if it's necessary and come back to cause some more IL-22-mediated inflammation to try to help get the problem resolved all happy like. But it has also been shown, by the group discussed in the paper, that this local inflammation can happen even without antigen-capable T-cells in a model of dermal delayed-type hypersensitivity.

And, wait!, if that isn't enough: it gets weird, too. In vitro studies have shown that restimulating TH17 cells with IL-23 can cause them to morph into TH1-like cells as characterized by IFNg (interferon gamma) production. Does this mean that TH17 cells themselves aren't a stable phenotype? Are they an evolutionary leftover that can segue into TH1 when necessary? Or are there unique TH17 inflammatory pathways that don't overlap much with the TH1 and TH2 pathways?

Either I don't know enough (very plausible), or we need more experiments!

Note: This paper was kind of odd in that it was a broad summary of current knowledge focusing on the work in 1 specific paper by authors not involved in that paper. Maybe it's a Nature thing. Also, note that I've come to the conclusion that "Not-So-Mad Science Blah Blah Blah" in unnecessary in research blogging. The icon is enough.

Yeonseok Chung, & Chen Dong (2009). Don't leave home without it: the IL-23 visa to TH-17 cells Nature Immunology, 10 (3), 236-238

Not-So-Mad Science: IL-13 vs. IL-4 In The Battle For Asthma!

(Previous asthma research-blogging here)

Marsha Wills-Karp, Jackie Luyimbazi, Xueying Xu, Brian Schofield, Tamlyn Y. Neben, Christopher L. Karp, Debra D. Donaldson (1998). Interleukin-13: Central Mediator of Allergic Asthma Science, 282, 2258-2261

Abstract:
The worldwide incidence, morbidity, and mortality of allergic asthma are increasing. The pathophysiological features of allergic asthma are thought to result from the aberrant expansion of CD4(+) T cells producing the type 2 cytokines interleukin-4 (IL-4) and IL-5, although a necessary role for these cytokines in allergic asthma has not been demonstrable. The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma. IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.

There are many morbidly fascinating pathological changes associated with onset and clinical asthma. To wit, these include eosinophilia, mucus overproduction, mast cell/other inflammatory cell airway infiltration, and increased smooth muscle. There may also be scarring of the airways.

From what I currently understand about it, airway hypersensitivity generally happens after immune effector cells have infiltrated the underlying airway tissues. When these effector cells, which can include allergen-specific T-cells, mast cells, eosinophils, basophils, and even macrophages, are activated by an irritant (the allergen) they more or less cut loose and let wild with the localized inflammation. The localized inflammation, in turn, leads to more immune cell infiltration over time and concurrently the tissue itself undergoes histopathologically apparent changes, including thickening of the base layer of smooth muscle.

For example of immune effector cells getting activated, let's consider the most dramatic case: the mast cell. Mast cells are a type of white blood cell that expresses Fc receptors for IgE (IgE is the immunoglobulin most associated with allergic and anti-parasite responses) on it's surface. The Fc-bound IgE act as allergen-specific receptors that, when bound to their ligand, send a signal into the cell to degranulate. Mast cells store relatively massive amounts of inflammatory cytokines and peptides in large granules (e.g., histamines, prostaglandins, and leukotrienes) and they can, effectively, disgorge them all at once. This can lead to a very rapid spike in the systemic concentration of inflammatory effector molecules and subsequently extremely rapid onset of asthmatic symptoms. The same process is at work in acute food allergies.

Figure A: The mast cell is the one with the big lumpy nucleus in the center. The black dots surrounding it are granules packed with inflammatory molecules, just waiting to be released and wreak havoc. Those other 2 cells to the right are lymphocytes (according to the original caption on this TEM).

But what inflammatory molecules are required to invoke and/or sustain a hypersensitive airway response?

This paper examined the role of IL-13 in allergic asthma. According to Janeway's Immunobiology, IL-13 is involved in the differentiation of naive CD4+ T-cells into TH2 cells, which have been shown to be more involved in allergy than TH1 cells. IL-13 is also secreted by TH2 cells, apparently, and has been shown to have a direct effect on airway epithelial cells by which their proliferation in increased and differentiation into goblet cells (goblet cell metaplasia) is increased, which in turn leads to the increased mucus production seen in allergy and asthma. And when your organs are infected with multicellular parasites, IL-13 is there to help the organs make the changes they need to get rid of those parasites. And as if that weren't enough, IL-13 also increases smooth muscle contractility.

But IL-13 doesn't really do anything without the context of a TH2 immune response. Th2 cells are characterized by secretion of IL-4, and it should be noted that IL-4 and IL-13 share a subunit in their receptors.

Figure B: The left column has a normal lung biopsy (top) and a normal airway (bottom) from a Tbet+/+ mouse. The right column has the same measurements, but showing airway inflammation with lymphocyte and eosinophil infiltration (top) and remodeled airway with increased collagen (bottom) from a Tbet-/- mouse. The picture is blurry because I took it with my phone. It is from Janeway's Immunobiology, 7th ed., page 575. Tbet is a transcription factor that is necessary for the development of TH1 cells, so its abscence will invariably result in a TH2 inflammatory response (right column). Tbet is analogous to GATA3 in TH2 cells.

Allow me to explain T-cell differentiation really briefly:

1) Naive T-cells arrive in thymus.
2) Naive T-cells have to decide whether or not to be CD4+ or CD8+, which will result in being able to recognize MHCII or MHCI, respectively.
3) CD4+ T-cells get stimulated by DCs or stuff, and the resulting cytokine mileau determines whether they become TH1, TH2, Treg (also refered to as TH3), or TH17. They can also become memory T-cells of any of those variety later on in. Respectively, these cell types are characterized by secretion of IFNg, IL-4/IL-5, IL-10, and IL-17.
4) TH1, TH2, Treg, and TH17 all more or less have distinct biological roles, although the cytokine soup that gives rise to different types is messy (e.g., IL-2 just drives T-cell proliferation irrespective of subset) and often overlaps, and they'll even compete against each other (IL-12 drives TH1 proliferation but inhibits TH2 proliferation while IL-4 does the same for TH2 cells).

So, anyway, the group behind this paper found that while IL-4 is sufficient to initiate asthmatic events, IL-13 is required for the development of the airway hypersensitivity response (AHR). They used the standard ovalbumin (OVA) induced model of AHR and found that blocking IL-13 with an neutralizing fusion protein prevents the development of AHR. Apparently blocking IL-13 in mice who already have AHR results in their measures of AHR decreasing (specifically goblet cell metaplasia and mucus production). However, with all of this, blocking IL-13 had no effect whatsoever on net circulating IgE or eosinophilia.

These findings prove that IL-13 has a significant role in asthma. But they also imply that IL-13 does not play this role through classical allergy pathways, as IL-13 is found to be elevated in patients with both allergic and non-allergic asthma. This is further supported by the group's finding that daily intratracheally administration of IL-13 is sufficient to induce asthmatic pathology even in the abscence of antigen sensitization.

What I wonder about here is: how does it make biological sense for a molecule involved heavily in the production of allergen-specific TH2 cells to also operate completely independently of that cellular phenotype?

But what is important to human health is that this paper demonstrates that adminstration of IL-13 agonists or blockers may be of great therapeutic value to human asthmatics. This paper is 11 years old, and I don't currently know if anything has come of their findings, but still, it'd be cool if this really did have therapeutic value because, as my last post on asthma discussed (link up top), inhaled acute anti-inflammatories may only be getting to the pieces of lung that need it least (because they're the pieces that can still pump air, and if reacting tissue isn't pumping tidal volume, how can inhaled medicine get to it?). If this could be used daily as a preventative, I think it could greatly improve the quality of life for asthmatics everywhere.