Background: NLRP3 inflammasomes regulate caspase-1-dependent IL-1␤ release and pyroptotic death in dendritic cells (DC) and macrophages. Results: Caspase-8 mediates IL-1␤ production and apoptosis by NLRP3 inflammasomes in caspase-1-deficient DC and facilitates pyroptosis in wild type DC. Conclusion: Caspase-8 is activated within NLRP3 inflammasome signaling platforms. Significance: In addition to caspase-1, NLRP3 inflammasomes engage caspase-8 as an important effector of innate immune signaling responses.

The identification of non-canonical (caspase-1 independent) pathways for IL-1β production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We found a heretofore unappreciated role for caspase-8 as a major pathway for IL-1β processing and release in murine bone marrow-derived dendritic cells (BMDC) co-stimulated with TLR4 agonists and pro-apoptotic chemotherapeutic agents such as doxorubicin (Dox) or staurosporine (STS). The ability of Dox to stimulate release of mature (17 kDa) IL-1β was nearly equivalent in wild-type (WT) BMDC, Casp1−/− Casp11−/− BMDC, WT BMDC treated with the caspase-1 inhibitor YVAD, and BMDC lacking the inflammasome regulators ASC, NLRP3, or NLRC4. Notably, Dox-induced production of mature IL-1β was temporally correlated with caspase-8 activation in WT cells and greatly suppressed in Casp8−/−Rip3−/− or Trif−/− BMDC, as well as in WT BMDC treated with the caspase-8 inhibitor, IETD. Similarly, STS stimulated robust IL-1β processing and release in Casp1−/−Casp11−/− BMDC that was IETD-sensitive. These data suggest that TLR4/TRIF activation induce assembly of caspase-8-based signaling complexes that become licensed as IL-1β converting enzymes in response to Dox and STS. The responses were temporally correlated with downregulation of cIAP1 suggesting suppressive roles for this and likely other Inhibitor of Apoptosis Proteins (IAPs) on the stability and/or proteolytic activity of the caspase-8 platforms. Thus, pro-apoptotic chemotherapeutic agents stimulate the caspase-8-mediated processing and release of IL-1β, implicating direct effects of such drugs on a non-canonical inflammatory cascade that may modulate immune responses in tumor microenvironments.

Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The Vmax (at 25 °C) for R194C is comparable to that of WT; while those of L311V, R396C, R396H and K246E are considerably reduced and affinity for adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity; whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 °C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60°C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 °C and after incubation at 37 °C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme’s structure and/or native conformation which are required for catalytic function.