Monocytes Take Their Own Path to IL-1β

Generally, two signals are required to activate the NLRP3 inflammasome. In this issue of Immunity, Hornung and colleagues (2016) describe an alternative inflammasome activation pathway in human monocytes triggering activation of the NLRP3 inflammasome in response to a single stimulus. Generally, two signals are required to activate the NLRP3 inflammasome. In this issue of Immunity, Hornung and colleagues (2016) describe an alternative inflammasome activation pathway in human monocytes triggering activation of the NLRP3 inflammasome in response to a single stimulus. The cytokine interleukin-1β (IL-1β) is critical for host defense against a wide variety of pathogens. Aberrant production of IL-1β is also pathological in many inflammatory diseases including atherosclerosis, Alzheimer's disease, pulmonary fibrosis, and diabetes. IL-1β is secreted as a result of inflammasome formation and caspase-1 activation, and requires caspase-1-mediated proteolytic maturation of pro-IL-1β to IL-1β and extrusion by an unknown mechanism. Activation of the nucleotide-binding domain leucine-rich repeat gene family-3 (NLRP3) inflammasome is classically considered a two-step process requiring both priming and activation (Figure 1) (Gurung et al., 2015Gurung P. Lukens J.R. Kanneganti T.-D. Trends Mol. Med. 2015; 21: 193-201Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). NF-κB-dependent priming is necessary for pro-IL-1β synthesis and NLRP3 upregulation, but priming also involves non-transcriptional changes such as post-translational modifications to NLRP3 and the adaptor molecule ASC. Once licensed through priming, NLRP3 activation requires a secondary stimulus, such as the ionophore nigericin. Although NLRP3 can be activated by many distinct stimuli, all known NLRP3 agonists require a K+ efflux. Whether K+ efflux is sufficient for activation, or whether events downstream of cation flux, such as disruption of mitochondrial integrity, are also required remains unclear (Gurung et al., 2015Gurung P. Lukens J.R. Kanneganti T.-D. Trends Mol. Med. 2015; 21: 193-201Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). It has long been known that human monocytes are unique in that they secrete IL-1β in response to a single stimulation with the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); however, the mechanism by which this occurs was unknown. In this issue of Immunity, Gaidt et al., 2016Gaidt M.M. Ebert T.S. Chauhan D. Schmidt T. Schmid-Burgk J.L. Rapino F. Robertson A.A.B. Cooper M.A. Graf T. Hornung V. Immunity. 2016; 44 (this issue): 833-846Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar describe an alternative NLRP3 inflammasome activation pathway with distinct requirements from canonical NLRP3 activation. Gaidt et al. demonstrate a potentially species-specific IL-1β secretory response of human and porcine monocytes stimulated with LPS alone, which was not detected in murine monocytes. It is unclear whether murine monocytes are truly incapable of LPS-induced IL-1β secretion, or whether murine LPS-induced IL-1β production was simply below the limit of detection of the experimental system used. Intriguingly, although inflammasome activation is considered to be a terminal event due to the induction of pyroptosis by caspase-1, LPS treatment alone did not drive pyroptotic cell death despite causing capase-1 activation and IL-1β secretion. These findings are in opposition with the theory that IL-1β release accompanies pyroptosis and provides evidence of a regulated IL-1β secretion mechanism preceding cell death. To explore IL-1β secretion pathways in human monocytes, the authors used an innovative approach to genetically manipulate human monocyte-like cells. They performed targeted gene ablations using CRISPR/Cas9 in an immortalized β-estradiol-inducible C/EBPα transgenic human B cell line (BLaER1). Transdifferentiating BLaER1 cells with β-estradiol, interleukin-3 (IL-3), and macrophage colony-stimulating factor (M-CSF) caused them to adopt a monocytic phenotype. Like primary monocytes, transdifferentiated BLaER1 cells secreted IL-1β following LPS stimulation in the absence of a second stimulus. Curiously, other human monocytic cells lines, such as THP-1 cells, did not activate the alternative inflammasome in response to LPS alone. BLaER1 monocyte-like cells also activated caspase-1 with nigericin treatment alone without requiring a priming stimulus (e.g., LPS or Pam3CSK4), suggesting that the transdifferentiation process might accomplish non-transcriptional priming requirements, but this requires further exploration. Cytosolic recognition of LPS by caspase-4 and -5 in humans forms a non-canonical inflammasome, which indirectly activates the NLRP3 inflammasome (Figure 1) (Shi et al., 2014Shi J. Zhao Y. Wang Y. Gao W. Ding J. Li P. Hu L. Shao F. Nature. 2014; 514: 187-192Crossref PubMed Scopus (46) Google Scholar), and a recent study suggests that caspase-4 and -5 might mediate LPS-induced IL-1β secretion by human monocytes (Viganò et al., 2015Viganò E. Diamond C.E. Spreafico R. Balachander A. Sobota R.M. Mortellaro A. Nat. Commun. 2015; 6: 8761Crossref PubMed Scopus (215) Google Scholar). In contrast, Gaidt et al. found that CRISPR/Cas9 ablation of caspase-4 and -5 in BLaER1 cells did not affect LPS-induced IL-1β secretion suggesting that non-canonical inflammasome activation was not involved. Despite this, all subsequent studies were performed in CASP4−/− BLaER1 cells to control for this possible source of interference. Additionally, because the propensity of monocytes to secrete IL-1β in response to LPS alone was previously attributed to ATP release and autocrine or paracrine P2X7R activation (Netea et al., 2009Netea M.G. Nold-Petry C.A. Nold M.F. Joosten L.A. Opitz B. van der Meer J.H. van de Veerdonk F.L. Ferwerda G. Heinhuis B. Devesa I. et al.Blood. 2009; 113: 2324-2335Crossref PubMed Scopus (601) Google Scholar, Piccini et al., 2008Piccini A. Carta S. Tassi S. Lasiglié D. Fossati G. Rubartelli A. Proc. Natl. Acad. Sci. USA. 2008; 105: 8067-8072Crossref PubMed Scopus (372) Google Scholar), the authors examined P2X7R-deficient BLaER1 cells and determined that IL-1β secretion was unaffected in their system in the absence of P2X7R (Gaidt et al., 2016Gaidt M.M. Ebert T.S. Chauhan D. Schmidt T. Schmid-Burgk J.L. Rapino F. Robertson A.A.B. Cooper M.A. Graf T. Hornung V. Immunity. 2016; 44 (this issue): 833-846Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Using CRISPR/Cas9-targeted genetic ablation in BLaER1 cells, Gaidt and colleagues confirmed that LPS-induced IL-1β maturation required the NLRP3 inflammasome as it was ablated in NLRP3−/−, ASC−/−, and CASP1−/− BLaER1 cells. TLR4 engages both the signaling adaptors MyD88 and TRIF, whereas other TLRs use only MyD88 with the exception of TLR3, which uses only TRIF. CRISPR/Cas9 mutagenesis revealed a requirement for TRIF downstream of TLR4 signaling in LPS-induced IL-1β secretion, explaining why the TLR2 agonist Pam3CSK4 did not induce alternative inflammasome activation. Intriguingly, treating cells with both Pam3CSK4 and the TLR3 agonist poly(I:C) to activate both MyD88 and TRIF-dependent signaling did not lead to IL-1β release, demonstrating that alternative inflammasome activation is specific to TLR4. Evaluation of known signaling cascades downstream of TRIF identified ripoptosome components being important regulators of the monocyte response to LPS. Necroptosis competent BLaER1 monocytes sufficient for receptor-interacting serine/threonine-protein kinase 3 (RIPK3) or mixed lineage kinase domain-like (MLKL), which were deficient for receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Fas-associated protein with death domain (FADD), or caspase-8 exhibited a strong necroptotic response accompanied by IL-1β release when stimulated with LPS, and this IL-1β processing required K+ efflux and therefore likely represents classical NLRP3 activation (Figure 1). However, BLaER1 cells, which were unable to necroptose (RIPK3−/− or MLKL−/−) and were also RIPK1-, FADD-, or caspase-8-deficient, demonstrated specific defects in alternative inflammasome activation (Figure 1). The caspase-8 requirement for alternative NLRP3 activation in BLaER1 cells required both catalytic activity and the capacity for autoproteolysis, suggesting that caspase-8-mediated cleavage of an unidentified target leads to NLRP3 inflammasome activation (Figure 1). Given the posited role of mitochondria in NLRP3 activation and the known role of mitochondrial caspase-8 in driving extrinsic apoptosis through BID activation and BAK and BAX pore formation, it is compelling to hypothesize that caspase-8 alters mitochondrial integrity to induce NLRP3 activation (Galluzzi et al., 2012Galluzzi L. Vitale I. Abrams J.M. Alnemri E.S. Baehrecke E.H. Blagosklonny M.V. Dawson T.M. Dawson V.L. El-Deiry W.S. Fulda S. et al.Cell Death Differ. 2012; 19: 107-120Crossref PubMed Scopus (1900) Google Scholar). The authors did not examine whether LPS alters mitochondrial morphology or function in human primary monocytes or BLaER1 cells, but this might be an interesting avenue for future research. A previous study found that BAK and BAX are dispensable for inflammasome activation in murine macrophages activated by canonical NLRP3 stimulation (Allam et al., 2014Allam R. Lawlor K.E. Yu E.C. Mildenhall A.L. Moujalled D.M. Lewis R.S. Ke F. Mason K.D. White M.J. Stacey K.J. et al.EMBO Rep. 2014; 15: 982-990Crossref PubMed Scopus (159) Google Scholar). However, it is clear that the requirements for canonical NLRP3 activation and alternative activation are distinct, and perhaps this divergence of signaling pathways extends to differential requirements for mitochondrial outer-membrane permeabilization. Additionally, the role of ripoptosome components has previously been studied in the context of inflammasome activation in mice, and murine macrophages double-deficient for RIPK3 and caspase-8 exhibit defects in inflammasome priming and NF-κB-dependent cytokine production in general (Allam et al., 2014Allam R. Lawlor K.E. Yu E.C. Mildenhall A.L. Moujalled D.M. Lewis R.S. Ke F. Mason K.D. White M.J. Stacey K.J. et al.EMBO Rep. 2014; 15: 982-990Crossref PubMed Scopus (159) Google Scholar, Gurung et al., 2014Gurung P. Anand P.K. Malireddi R.K. Vande Walle L. Van Opdenbosch N. Dillon C.P. Weinlich R. Green D.R. Lamkanfi M. Kanneganti T.D. J. Immunol. 2014; 192: 1835-1846Crossref PubMed Scopus (357) Google Scholar). However, because ripoptosome-deficient BLaER1 cells did not demonstrate a priming defect, it is possible that species-specific differences exist in the signaling adaptors and scaffolding utilized downstream of TLR4. Importantly, having genetically discerned the signaling pathway for alternative inflammasome activation, the authors then returned their studies to primary human monocytes and employed pharmacologic inhibitors of NLRP3, caspase-1, caspase-8, and RIPK3 to demonstrate that the same signaling pathway governing alternative NLRP3 activation in BLaER1 monocyte-like cells occurs in primary human monocytes. Because NLRP3 inflammasome activation traditionally requires K+ efflux, the authors examined whether high extracellular K+ affected LPS-induced IL-1β secretion. Surprisingly, the alternative inflammasome pathway did not require K+ efflux. The demonstration of a K+ efflux-independent NLRP3 activation pathway challenges the dogma that K+ flux is necessary and sufficient for NLRP3 activation and supports a model where events downstream of cation flux lead to activation. Additionally, alternative inflammasome activation neither causes cell death and lactate dehydrogenase (LDH) release nor leads to ASC pyroptosome formation. This supports the idea that ASC pyroptosome formation, and not caspase-1 activation alone, is required for pyroptosis. The authors did not examine cleavage and activation of Gasdermin D, which was recently identified as a key mediator of pyroptosis downstream of inflammatory caspase activation (Kayagaki et al., 2015Kayagaki N. Stowe I.B. Lee B.L. O'Rourke K. Anderson K. Warming S. Cuellar T. Haley B. Roose-Girma M. Phung Q.T. et al.Nature. 2015; 526: 666-671Crossref PubMed Scopus (1877) Google Scholar). Assessing whether alternative inflammasome activation leads to Gasdermin D cleavage would help determine whether ASC pyroptosomes are required for Gasdermin D activation and whether Gasdermin D proteolysis is sufficient for pyroptosis. Gaidt et al. have identified an additional pathway for NLRP3 activation, which diverges from canonical activation in the number of stimuli required, the induction and reliance on K+ efflux, and the ultimate cell fate. The discovery that TLR4 activation leads to RIPK1-, FADD-, and caspase-8-dependent activation of NLRP3 explains the previously enigmatic phenomenon of LPS alone-induced IL-1β secretion. Humans demonstrate high sensitivity to endotoxin when compared to other mammals, and although a number of mechanisms have been posited to explain this shock susceptibility, Gaidt et al. have added an additional possibility that endotoxemia in humans could lead circulating peripheral blood monocytes to produce IL-1β through alternative NLRP3 inflammasome activation. Certainly it will be important to address the role of alternative inflammasome activation by monocytes in vivo. Because studying the biological significance of the alternative inflammasome will be challenging to address in human systems, future studies might seek to utilize humanized mouse or pig models. Furthermore, it would be intriguing to determine whether endogenous TLR4 agonists are able to induce alternative inflammasome activation and whether this alternative pathway plays a pathogenic role in sterile inflammatory disorders and chronic inflammatory diseases. NIH grants R56 AI118719 (F.S.S.) and T32 GM007337 (E.I.E.) supported this work. Human Monocytes Engage an Alternative Inflammasome PathwayGaidt et al.ImmunityMarch 29, 2016In BriefHow human monocytes secrete processed IL-1β upon LPS challenge is unknown. Hornung and colleagues report that LPS triggers an alternative NLRP3 inflammasome pathway in human monocytes. Unlike classical NLRP3 inflammasome signaling, alternative inflammasome activation proceeds independently of potassium efflux, pyroptosome formation, and pyroptosis, while it engages TLR4-TRIF-RIPK1-FADD-CASP8 upstream of NLRP3. Full-Text PDF Open Archive