Editors' ChoiceInflammation

A20: Belt and braces

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Sci. Signal.  03 Feb 2015:
Vol. 8, Issue 362, pp. ec24
DOI: 10.1126/scisignal.aaa8161

The inflammatory cytokine interleukin-1β (IL-1β) is produced by cells in a two-step process. First, nuclear factor κB (NF-κB) signaling activated by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), results in the production of the pro-IL-1β protein. Second, cytosolic danger signals, such as ATP, stimulate the formation of multiprotein complexes (inflammasomes), which contain caspase-1. Proteolytic processing of pro-IL-1β by caspase-1 produces the mature form of the cytokine, which is secreted. A20 is a deubiquitinase that mediates feedback inhibition of NF-κB signaling. Duong et al. showed that A20 also directly inhibits NLRP3 inflammasome activity. Unlike macrophages from wild-type mice, macrophages from A20-deficient mice secreted IL-1β in response to the TLR4 ligand lipopolysaccharide (LPS) without the need for a second signal. Loss of NLRP3 components from the A20-deficient cells blocked IL-1β secretion, suggesting that NLRP3 was spontaneously active in these cells. This effect of loss of A20 depended on the kinase RIPK3, which has been implicated in NLRP3 activation, but is not a component of the inflammasome. Coimmunoprecipitation studies of LPS-treated wild-type macrophages showed that A20 interacted with pro-IL-1β in a complex that also contained RIPK3 and caspase-1. Activation of these cells with ATP enhanced these interactions. The pro-IL-1β–containing complex in LPS-treated wild-type macrophages also contained lysine 63 (K63)–linked and unanchored polyubiquitin chains. The extent of ubiquitylation of this complex was greater in A20-deficient cells than in wild-type cells and depended on RIPK3. Ubiquitylation of the pro-IL-1β–containing complex in A20-deficient cells was unaffected by the loss of NLRP3 components, and mass spectrometric analysis showed that pro-IL-1β was directly ubiquitylated at Lys133. Furthermore, studies of wild-type and a K133R mutant of pro-IL-1β in transfected cells showed that ubiquitylation of Lys133 promoted the processing of pro-IL-1β by caspase-1. Together, these data suggest that A20 restricts the ability of the NLRP3 inflammasome to process its substrate pro-IL-1β and thus suppresses inflammatory responses.

B. H. Duong, M. Onizawa, J. A. Oses-Prieto, R. Advincula, A. Burlingame, B. A. Malynn, A. Ma, A20 restricts ubiquitination of pro-interleukin-1β protein complexes and suppresses NLRP3 inflammasome activity. Immunity 42, 55–67 (2015). [PubMed]