Mystery Rays from Outer Space

A couple of years ago, Jürg Tschopp’s group showed that uric acid crystals acted as inflammatory agents (and probably, also as adjuvants) by stimulating the Nalp3 inflammasome.¹ A month ago, Richard Flavell’s group showed that alum adjuvant — also (sort of) crystalline — also acts through the Nalp3 inflammasome.² And now, a paper just out in PNAS says that crystalline silica causes silicosis by acting through, you’ll never guess, the Nalp3 inflammasome. ³ Could a trend be showing up?

I don’t know much about silicosis, other than the obvious stuff (it’s an inflammatory lung disease caused by inhaling crystalline silica) and to be honest I had never much wondered why silica would cause lung disease; I assumed that it’s toxic, caused cell damage because of direct cytotoxicty, and the cell damage led to an inflammatory response. It turns out that that’s partly right; silica is cytotoxic and does cause cell damage, but the cell damage per se is not the cause of the inflammation, because Nalp3-knockout mice still had the same amount of cell death, but didn’t have the inflammation.

Nalp3 is an intracellular sensor, which raises an obvious question⁴ about all these crystals: How do they get into the cell to stimulate the response? As far as I know, this is the first of the papers to look at this question, and the answer is a little disappointing in that it seems fairly simple: It’s just endocytosis.

… endocytosis of silica by macrophages is needed to activate the Nalp3 inflammasome in response to silica for the resultant processing and secretion of proinflammatory cytokines.

I was at least half expecting a much more complicated and exciting answer, but this does make sense.  (I don’t know, actually, if silica, alum, and uric acid crystals are about the same size, or if for some other reason endocytosis is not a plausible explanation for the other two.)  That still leaves the issue of how the crystals get out of the endosome and into the cytosol, but we do know that in macrophages and dendritic cells there’s a poorly-characterized pathway by which some substances — proteins that are cross-presented, in particular — can exit the endosome and gain access to the cytosol, so we’re not adding any new mysteries, anyway.

The next candidate is probably asbestos; at this point, I think it’s fairly likely that asbestosis is also mediated by the Nalp3 inflammasome.

The only one of these crystalline substances that’s physiological is uric acid — monosodium urate crystals, a natural adjuvant that’s believed to act as a danger signal for cell death. I wonder if all these things are mimicking MSU crystals, or if there’s some other reason they act through the same receptor.

1. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. 440, 237 - 241 (11 Jan 2006), doi:10.1038/nature04516[↩]
2. Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature  453, 1122-1126 doi:10.1038/nature06939[↩]
3. Cassel, S.L., Eisenbarth, S.C., Iyer, S.S., Sadler, J.J., Colegio, O.R., Tephly, L.A., Carter, A.B., Rothman, P.B., Flavell, R.A., Sutterwala, F.S. (2008). The Nalp3 inflammasome is essential for the development of silicosis. Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0803933105[↩]
4. Brought up by Kay, last time I talked about it[↩]

Vaccination is one of the (if not the most) important medical advances in history. The problem today is that most of the easy diseases already have vaccines available, and now we’re trying to develop vaccines against the hard ones. Fortunately, I think we’re entering a new golden age of vaccine development, as we begin to understand why immunization works at the molecular level, to the point where we may soon be able to deliberately tweak them for optimal efficacy.

Back in the dark ages, when I was first working with vaccines,¹ adjuvants were a witches’ brew of newts’ eyes and frogspawn, and the ones that worked, just sort of … worked. No one really knew why. But around the time I backed away from vaccines, (partly because of this empirical adjuvant stuff) new theoretical frameworks were being developed that began to explain how and why adjuvants work, and now — some 20 years later — we are at the point where theory is moving solidly toward practice.

I’ve commented several times on the issue of immunodominance. T cell responses to antigens aren’t smoothly distributed over all the possible targets in the antigen; instead, a handful of targets get the lion’s share of the T cell response. Sometimes this is a good thing (for example, it’s a way of getting a screaming hot response to a target, instead of having a bunch of wimpy little responses); sometimes it’s bad (if it’s a moving target, as with rapidly-mutating viruses such as HIV, then your screaming hot response may be to a target that no longer exists, whereas having a bunch of targets at least nearly guarantees that you’ve got something to shoot at.)

In spite of its importance, though, the underlying mechanisms that drive immunodominance aren’t well understood. For example, one possible explanation is that the T cell that ends up becoming dominant, started out as the most abundant clone originally. A paper last year² (I talked about it here) supported that possibility, but a more recent study³ that I talked about earlier this week suggested that while clonal abundance is one factor, there must be other, equally important, influences on the response.

That fits with another paper that came out in May,⁴ looking at the effects of different adjuvants on the immune response. Of course this has been done many times in a quantitative way — which adjuvant gives the biggest response? — but Malherbe et al. asked the question qualitatively: What exactly happens to the T cell response? That is: We know that different adjuvants can cause higher or lower responses to an antigen; but are the different responses made up of the same CTL⁵, or do different adjuvants crank up different sets? Can we drive a T cell response that is qualitatively, as well as quantitatively, better?

I, for one, (and I think most of the field) would have said “No”; no matter what your adjuvant is, the response would be qualitatively the same. Why would one particular CTL precursor clone be stimulated better or worse by a particular adjuvant? That’s the answer that would be predicted from the first study, that suggested that immunodominance is determined mainly by the precursor frequency: You can’t really affect the precursor frequency (that’s set during thymic development), so no matter what you do with your antigen you should get the same relative response (even though the total response may be higher or lower, it would contain the same proportion of T cell clones).

In fact, that’s not what happens. Malherbe et al. compared five different adjuvants, mixed with the same antigen. The adjuvants are known to act through different mechanisms. (That is, while they all act by stimulating innate immune recognition molecules, they stimulate different innate receptors — different TLR molecules, or [as we now know⁶ ] pattern recognition receptors that are different from TLRs altogether.) Then they assessed the subsequent immune response by comparing the immunodominance hierarchies that came out of the immunization. The different adjuvants drove expansion of different T cell clones, so that the response was qualitatively different.

In particular, adjuvants drove expansion of higher-affinity clones:

…adjuvants regulate clonal composition by using a mechanism that alters initial TCR-based selection thresholds and that relies most heavily on blocking the propagation of antigen-specific clonotypes expressing low-affinity TCR. … Thus, adjuvant formulation can modify the TCR-based selection threshold that regulates Th cell clonal composition in response to protein vaccination.

How adjuvants do this remains unknown. It wasn’t related to the antigen dose (which has previously been shown to affect the TcR affinity). Possibilities include differential dendritic cell maturation, altering local antigen contentration (the “depot” effect that has been the classic explanation for alum’s mechanism of action — though that explanation is at least partly rendered obsolete by the recent paper⁷ from Richard Flavell’s group), and direct stimulation of T cell clones — but who knows.

Assuming this holds up for different antigens (they’ve only looked at one, so far) the key thing, in clinical terms, is that it’s possible to alter immunodominance without changing the antigen. We need to understand how this works, because it may be a much simpler way of improving immune responses than altering the antigen itself.

1. It looks as if I may be doing so again; our proposal for a Vaccine Center here has been funded, at least for a few years; although I’m only a small part of the group[↩]
2. Naive CD4(+) T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude. Moon JJ, Chu HH, Pepper M, McSorley SJ, Jameson SC, Kedl RM, Jenkins MK. Immunity. 2007 Aug;27(2):203-13.[↩]
3. Obar, J., Khanna, K., LeFrancois, L. (2008). Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection. Immunity, 28(6), 859-869. DOI: 10.1016/j.immuni.2008.04.010[↩]
4. Malherbe, L., Mark, L., Fazilleau, N., McHeyzer-Williams, L., McHeyzer-Williams, M. (2008). Vaccine Adjuvants Alter TCR-Based Selection Thresholds. Immunity, 28(5), 698-709. DOI: 10.1016/j.immuni.2008.03.014

   Commentary at:
   Immunity 28:602-604 (16 May 2008) doi:10.1016/j.immuni.2008.04.008
   Preview: Taking a Toll Road to Better Vaccines
   Sharon Celeste Morley and Paul M. Allen[↩]

5. CTL: Cytotoxic T lymphocytes[↩]
6. Eisenbarth, S.C., Colegio, O.R., O'Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature DOI: 10.1038/nature06939[↩]
7. Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature. DOI: 10.1038/nature06939[↩]

Inflammasomes (From Yu et al, 2005)1

We’ve known for quite a while now how adjuvants work. Adjuvants (the components of vaccines that cause an immune response to start up, but that are not themselves the target of the immune response) trigger the parts of the innate immune system that normally identify microbial patterns, so that the immune system becomes aware that there’s a dangerous situation on its hands. (I have a much longer explanation here.)

It was a little annoying, though, that the most important adjuvant — alum, the adjuvant most commonly used for human vaccines — didn’t fit into this explanation. We didn’t know what innate triggers alum tickled to drive immune responses.

A month or two ago, I talked about a paper² that claimed to answer that long-standing question. Their answer is that alum works through damaging tissue; damaged tissue releases a danger signal, uric acid, and according to Kool and the Gang² that’s how alum drives immune responses. It’s an interesting suggestion, but I didn’t buy it:

I’m not entirely convinced that this is the whole, or even the main, story. … simple experience says that while vaccines sting, you don’t expect any kind of large-scale necrosis in your injected arm afterward - no more than you’d get from a modest bruise, which isn’t enough to trigger the kind of adjuvant effects we see with alum.

A paper that’s just become available in advance online status³ backs up my skepticism.

This is from Richard Flavell’s group at Yale. They determined that alum activity, at physiological doses, requires an intracellular receptor, Nalp3, that’s a member of the NLR family. NLRs (”Nod-like receptors”) are conceptual parallels to TLRs (”Toll-like receptors”); TLRs and NLRs are receptors for danger signals, and Nalp3 in particular is a receptor for uric acid, among other things. ⁴

Strictly speaking, uric acid itself does not act as a danger signal. Uric acid is a normal component of extracellular tissues, and if it was inflammatory we’d be in perpetual agony. When dying cells release their own stores of uric acid, though, the surrounding fluid becomes overloaded, and uric acid precipitates out as monosodium urate (MSU) crystals. It’s these MSU crystals that are actually inflammatory. Flavell’s insight was that MSU crystals might be conceptually siimilar to alum — another insoluble, particulate adjuvant — and so the two might act through the same pathway.

Sure enough, knockout mice without Nalp3 (or without other components of the Nalp3 reconition particle) failed to respond to alum (or to uric acid), whereas knockouts for a different NLR member did just fine. The knockouts responded normally when a different adjuvant was used.

Thus, by eliminating signalling through the Nalp3 inflammasome, we have eliminated one critical pathway used by alum to initiate humoral and cellular immunity. In doing so, aluminium hydroxide adjuvants ‘hijack’ an innate immune pathway that is exquisitely sensitive to cellular damage, perhaps as a result of the similarity to MSU in its physical structure.

The effect is pretty striking, actually (for example, the figure to the right).

The previous paper I talked about (Kool et al)² also pinned alum into the uric acid pathway, but reached the conclusion that alum works through uric acid, rather than parallel to it, by causing tissue damage. Flavell’s group agree that this can happen, but disagree that it’s the normal mode of action:

In vitro, alum induced cell death at very high doses … in WT macrophages and in macrophages deficient in Nalp3 and Caspase-1 (Fig. 3b); however, the induction of IL-1beta by alum did not depend on the presence of MSU because the addition of uricase, which degrades MSU crystals and prevents the induction of IL-1beta (ref. 10), had no effect on IL-1beta production in response to LPS and alum (Fig. 3c).

I’m altogether happier with this explanation.

One interesting question that leaps to my mind — now we know the genes involved in alum recognition — is whether natural variants in these genes exist (I’m sure they do) and whether such variants correlate with vaccine responses in humans. Could we predict whether someone only needs a single dose of vaccine to be protected compared to her cousin who needs three doses? Are there people who lack components of this altogether — like the various TLR3 mutants I talked about earlier — and what happens to their vaccine response?

1. Yu JW, Wu J, Zhang Z, Datta P, Ibrahimi I, Taniguchi S, Sagara J, Fernandes-Alnemri T, Alnemri ES (2006) Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization. Cell Death Differ 13:236–249. doi:10.1038/sj.cdd.4401734[↩]
2. Kool, M., Soullie, T., van Nimwegen, M., Willart, M.A., Muskens, F., Jung, S., Hoogsteden, H.C., Hammad, H., Lambrecht, B.N. (2008). Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. Journal of Experimental Medicine DOI: 10.1084/jem.20071087[↩][↩][↩]
3. Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature DOI: 10.1038/nature06939 [↩]
4. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440:237-241. doi:10.1038/nature06939[↩]

My first foray into research, when I started grad school, was to work on a vaccine against bovine adenovirus type 3.¹ From a technical viewpoint, it was a great introduction to research; I got to do classical virology, animal work, tissue culture, protein purification, all kinds of immunology, and so on. Still, I ended up unenthusiastic about vaccinology (though not about vaccines, which I regard as one of the most important benefits to civilization in history). The problem was that so much of vaccine research seemed purely empirical, with no solid theory underlying it. The way to design a new vaccine was just to try a whole bunch of different things (sometimes applying rules of thumb) and see what worked.

To some extent that’s still true — there’s a great deal we still don’t understand about the immune system, and predicting how to drive a safe, protective response still has a lot of guesswork to it. ² At the time, though, the most frustrating aspect of the field for me was adjuvants.

Adjuvants are factors that you include with your antigen, in order to drive a potent immune response. In general, the more pure an antigen is, the worse the immune response to it. Adjuvants allow you to provide a clean, defined antigen, without losing the immunogenicity of the filthy natural antigen.

The problem was that no one knew how adjuvants worked. They just … worked. There were a myriad of choices (for animals; in the US and Canada there’s only one adjuvant, alum, that’s licensed for humans), and they all mostly worked, and sometimes one worked better and sometimes another worked better, or differently; but there was no understanding of how, or why. Sometimes toe of newt was the best choice, and sometimes you were better off with eye of toad, and it depended on the phase of the moon and on which malign vapours were influencing your system.

Had I but known, of course, just about the time I switched out of vaccines and into other aspects of viral immunity, Charlie Janeway was offering up a grand unified theory of adjuvants³ which has for the most part proven triumphantly true. (I talked about it here.) He suggested that the immune system normally initiates a response when it recognizes conserved features of pathogens, and that adjuvants work because they mimic these conserved pathogen-associated molecular patterns. (Polly Matzinger also proposed a related model, in which immune responses start because cells are damaged — the danger hypothesis.) Since then, many of the pathogen-associated patterns have been identified, and many of the pattern receptors have been identified; adjuvants are no longer magic, they’re science.

Well, all except for one: Alum, the most important one of all (because it’s the main adjuvant used for human vaccines). We had no real idea how that works, because it looks nothing like any plausible pathogen pattern.

A paper in J Exp Med⁴ now argues that alum’s adjuvant activity comes from uric acid. As it happens, this is less related to Janeway’s hypothesis, and is closer to Matzinger’s. Uric acid is released by dying or damaged cells, and is a powerful natural adjuvant⁵ — it’s an indicator to the immune system that cells are being damaged in the vicinity, meaning that there is “danger” nearby.

I’m not entirely convinced that this is the whole, or even the main, story. The implication is that alum acts by damaging cells. The authors say that “alum has been shown to induce a considerable degree of necrosis”. That may be true with the intraperitoneal injection model they used, but alum-adjuvanted vaccines in people are more often given intramuscularly, and I don’t know that alum is all that nasty in that context.⁶ After all, the reason alum is approved for use in humans is that it is so innocuous. And simple experience says that while vaccines sting, you don’t expect any kind of large-scale necrosis in your injected arm afterward — no more than you’d get from a modest bruise, which isn’t enough to trigger the kind of adjuvant effects we see with alum. Perhaps there is a small release of uric acid effect, and alum somehow amplifies the effect (perhaps by facilitating uric acid crystallization, which is essential for its adjuvant activity). Or perhaps uricase is important in intraperitoneal inject, but is less so in more clinically-relevant injections. I don’t know.

Still, it’s nice to see that adjuvant activities, nowadays, can actually be tested within well-defined theoretical contexts. That’s just a huge advance since the days when I had to play with them.

1. York, I., and Thorsen, J. (1992). Evaluation of a subunit vaccine for bovine adenovirus type 3. American journal of veterinary research 53, 180-183.[↩]
2. Partly, of course, because most of the easy targets for vaccines, where we could predict protective responses, are already out there, and now we’re working on the hard cases like malaria and HIV.[↩]
3. Approaching the asymptote? Evolution and revolution in immunology. Janeway, C.A.Jr.. Cold Spring Harb. Symp. Quant. Biol. 54, 1-13 (1989) [↩]
4. Kool, M., Soullie, T., van Nimwegen, M., Willart, M.A., Muskens, F., Jung, S., Hoogsteden, H.C., Hammad, H., Lambrecht, B.N. (2008). Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. Journal of Experimental Medicine DOI: 10.1084/jem.20071087[↩]
5. Shi, Y., Evans, J. E., and Rock, K. L. (2003). Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425, 516-521.[↩]
6. A quick and superficial scan through PubMed doesn’t turn up much support for the statement either , for what that’s worth.[↩]