Inflammasome immortality

Immune system has many ways to detect the presence of virulence factors. One such system is an assembly of the super-complex called inflammasome. For its proper activity this super-complex requires close association of several components: NALP3, ASC and pro-Caspase 1. Depending on initial trigger, NALP3 can be replaced with NLRP1, AIM2, NLRC4 in inflammasome super-complex. Once formed, inflammasome cleaves pro-IL-1 or pro-IL-18 into mature active proteins IL-1beta and IL-18.

Earlier studies have shown that inflammasome assembly and function occurs in the cytoplasm. Now, two new papers published in Nature Immunology reveal that inflammasome continues to function as a secreted, extracellular template and amplifies local IL-1 production and recruitment of granulocytes.

I am going to discuss the results of several principal experiments from these studies.

In the first paper (1), the authors generated macrophage line expressing ASC molecules fused to fluorescent protein (ASC-FP). In resting state, ASC-FP were evenly distributed in cytoplasm. Upon inflammasome activation, however, ASC-FP formed oligomers. Interestingly, the authors detected substantial amount of ASC-FP extracellularly. This release of ASC was associated with cell death and requires caspase 1 activity. Treatment of LPS-primed ASC-KO macrophage cytosol with recombinant ASC showed that recombinant ASC had pro-caspase-1 and pro-IL-1 processing activity, while LPS+ATP combination did not. These results indicated that exogenously added ASC formed super-complexes with endogenous NALP3 and interacted with pro-caspase 1.

To directly observe this phenomenon, the authors used red fluorescent tagged ASC-mCherry construct. They found that (a) ASC-mCherry was phagocytosed and released inside macrophage's cytosol after lysosomal membrane destabilization and it formed oligomers, (b) as a result, an exogenous ASC-mCherry acted as a danger signal for LPS-primed macrophages inducing IL-1beta secretion, similar to silica or nigericin.

More importantly, using two different fluorescent tagged ASC recombinant proteins, ASC-mCherry (red) and ASC-mCerulean (blue), the authors observed that exogenously added ASC attracted endogenous ASC in super-complexes, acted as a template for further oligomerization.

Finally, the authors conducted several in vivo experiments with recombinant ASC and found that injection of exogenous ASC oligomers induced local inflammatory granulocytes recruitment independent of NALP3, but IL-1 receptor dependent manner (this effect was enhanced in presence of anti-ASC antibody or in presence of serum derived from autoimmune mouse strain). Of note, there is opposite results regarding exogenous ASC ability to induce IL-1beta secretion from ASC-KO macrophages (compare Fig .3f and Fig. 3g versus Fig 5e). Either there is reporting error or in vitro bone marrow derived macrophages behave differently from ex vivo inflammatory macrophages.

In second paper (2), the authors also confirmed that inflammasome components were detected in supernatant within 15 min of macrophage activation and they formed oligomers. They also found that addition of recombinant ASC oligomers to LPS-primed ASC-KO macrophage cell line induced IL-1beta secretion (however this required presence of ATP or NALP3, a slightly different result from the first paper). The difference could be due to use of primary macrophages versus macrophage cell line (or immortalized macrophages) in different series of experiments, am obvious limitation. Indeed, comparison of IL-1beta secretion results in Fig. 3J versus Fig. 4B, clearly indicates that dependency on ATP or NALP3 is a function of difference in cell types or difference in dose of recombinant ASC oligomers.

In summary, these papers showed that during inflammasome induced pyroptosis, dying cells release active complexes of inflammasomes that are able to directly process extracellular pro-IL-1 locally or alternatively these oligomers internalized by local macrophages and act as danger signals in propagating inflammation. Since inflammasome-related mutations are linked to several sterile inflammatory diseases, improved understanding of inflammasome biology could yield more targeted therapeutics.

David