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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?
- 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[↩]
- 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[↩][↩][↩]
- 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 [↩]
- 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[↩]
This is all very nice, but how do the Alum crystals get to the inflammasome? Unlike the TLRs, the NLRs are cytoplasmic and I have always liked the idea that NALP-based recognition is mainly for endogenous stimuli. I must say that Jurg was also rather vague on this issue in his gout paper (sorry Jurg, but I guess that you are not reading this anyway, or are you?)
Kay, as always, the short answer is likely “Don’t know”. For uric acid — I’ve seen micrographs (not published, I think) of monosodium urate crystals mixed with dendritic cells. The MSU crystals are long, needle-like things and they actually seem to penetrate the cells, sticking right through them. Bizarrely this doesn’t seem to kill the cells. I find it hard to believe that this is actually what is happening — maybe it’s some kind of illusion, and the crystals end exactly at the membrane; or maybe the cells really are dead, though assays don’t indicate it — but it’s at least conceivable that it’s real and the MSU can physically enter the cytosol.
I’ve never seen alum crystals and I don’t know if they look anything like this.
[…] 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 […]
[…] 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 […]
Hi, yes, there is published this and the results are important:
Arthritis Rheum. 2008 Mar;58(3):888-94.
Gene polymorphisms in the NALP3 inflammasome are associated with interleukin-1 production and severe inflammation: relation to common inflammatory diseases?Verma D, Lerm M, Blomgran Julinder R, Eriksson P, Söderkvist P, Särndahl E.
… RESULTS: Mutation analysis of the patient’s genes encoding NALP3, ASC, and TUCAN revealed variations in the NLRP3 (Q705K) and CARD-8 (C10X) genes. The allele frequencies of these single-nucleotide polymorphisms (SNPs) in the population were 6.5% and 34%, respectively. .. CONCLUSION: Our results indicate that the patient’s symptoms were due to elevated levels of IL-1beta, since treatment with anakinra effectively abolished the symptoms. The compound SNPs may explain the increased IL-1beta levels and inflammatory symptoms observed, but further studies are needed to reveal a functional relationship. The prevalence of the polymorphisms (4% of the population carry both SNPs) in the general population may suggest a genetic predisposition for common inflammatory disorders.
[…] A couple of years ago1 it was discovered that the natural adjuvant uric acid stimulates immunity through an intracellular sensor called the Nalp3 inflammasome. Last year, a bunch of groups showed the the commonly-used adjuvant alum — also kind of crystalline — acts through the Nalp3 inflammasome. 2 Then it turned out that silca causes the inflammation associated with silicosis via, right, the Nalp3 inflammasome. 3 (I talked about this in several places, including here.) […]
it has been mentioned that alum activates the the innate immune system, my question is, how is a better immune response against an Ag conjugated with alum generated?
(forgive me if my question sounds elementary, i am a student of basic immunology)
i’m not as scientific as i’d like to be but right off the bat, uric acid signaling our immune system is just wrong. for uric acid to trigger something is just really physically painful. thanks for shedding light on this. i always enjoy your posts and expanding my knowledge on health and science.