ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF.

ZBP1 is widely recognized as a mediator of cell death for its role in initiating necroptotic, apoptotic, and pyroptotic cell death pathways in response to diverse pathogenic infection. Herein, we characterize an unanticipated role for ZBP1 in promoting inflammatory responses to bacterial lipopolysaccharide (LPS) or double-stranded RNA (dsRNA). In response to both stimuli, ZBP1 promotes the timely delivery of RIPK1 to the Toll-like receptor (TLR)3/4 adaptor TRIF and M1-ubiquitination of RIPK1, which sustains activation of inflammatory signaling cascades downstream of RIPK1. Strikingly, ZBP1-mediated regulation of these pathways is important in vivo, as Zbp1−/− mice exhibited resistance to LPS-induced septic shock, revealed by prolonged survival and delayed onset of hypothermia due to decreased inflammatory responses and subsequent cell death. Further findings revealed that ZBP1 promotes sustained inflammatory responses by mediating the kinetics of proinflammatory "TRIFosome" complex formation, thus having a profound impact downstream of TLR activation. Given the well-characterized role of ZBP1 as a viral sensor, our results exemplify previously unappreciated crosstalk between the pathways that regulate host responses to bacteria and viruses, with ZBP1 acting as a crucial bridge between the two.

LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

Flipping the Switch from Inflammation to Cell Death.

Multiple research groups have demonstrated that caspase-8 (CASP8)-mediated gasdermin D (GSDMD) cleavage drives pyroptotic cell death. Here, we discuss a novel role for the enzymatically inactive homolog of CASP8, the long isoform of cellular FLICE-like inhibitory protein (cFLIPL), in the regulation of this process. Specifically, cFLIP-deficiency provides a model in which to study the mechanisms regulating CASP8-mediated activation of cell death and inflammatory signaling.

Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection.

Cell death and inflammation are intimately linked during Yersinia infection. Pathogenic Yersinia inhibits the MAP kinase TGFß-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8-mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. We found that the Yersinia-driven IL-1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations are required to provide signal 1 and signal 2 for IL-1α/ß release. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1ß production. Our results uncover a form of caspase-8-mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir.

Neutrophils and macrophages drive TNF-induced lethality via TRIF/CD14-mediated responses.

TNF mediates a variety of biological processes including cellular proliferation, inflammatory responses, and cell death and is therefore associated with numerous pathologies including autoinflammatory diseases and septic shock. The inflammatory and cell death responses to TNF have been studied extensively downstream of TNF-R1 and are believed to rely on the formation of proinflammatory complex I and prodeath complex II, respectively. We recently identified a similar multimeric complex downstream of TLR4, termed the TRIFosome, that regulates inflammation and cell death in response to LPS or Yersinia pseudotuberculosis. We present evidence of a role for the TRIFosome downstream of TNF-R1, independent of TLR3 or TLR4 engagement. Specifically, TNF-induced cell death and inflammation in murine macrophages were driven by the TLR4 adaptor TRIF and the LPS co-receptor CD14, highlighting an important role for these proteins beyond TLR-mediated immune responses. Via immunoprecipitation and visualization of TRIF-specific puncta, we demonstrated TRIF- and CD14-dependent formation of prodeath and proinflammatory complexes in response to TNF. Extending these findings, in a murine TNF-induced sepsis model, TRIF and CD14 deficiency decreased systemic inflammation, reduced organ pathology, and improved survival. The outcome of TRIF activation was cell specific, because TNF-induced lethality was mediated by neutrophils and macrophages responding to TNF in a TRIF-dependent manner. Our findings suggest that in addition to their crucial role in TNF production, myeloid cells are central to TNF toxicity and position TRIF and CD14 as universal components of receptor-mediated immune responses.

ZBP1 promotes LPS-induced cell death and IL-1ß release via RHIM-mediated interactions with RIPK1.

Inflammation and cell death are closely linked arms of the host immune response to infection, which when carefully balanced ensure host survival. One example of this balance is the tightly regulated transition from TNFR1-associated pro-inflammatory complex I to pro-death complex II. By contrast, here we show that a TRIF-dependent complex containing FADD, RIPK1 and caspase-8 (that we have termed the TRIFosome) mediates cell death in response to Yersinia pseudotuberculosis and LPS. Furthermore, we show that constitutive binding between ZBP1 and RIPK1 is essential for the initiation of TRIFosome interactions, caspase-8-mediated cell death and inflammasome activation, thus positioning ZBP1 as an effector of cell death in the context of bacterial blockade of pro-inflammatory signaling. Additionally, our findings offer an alternative to the TNFR1-dependent model of complex II assembly, by demonstrating pro-death complex formation reliant on TRIF signaling.

cFLIPL protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation.

Cell death and inflammation are interdependent host responses to infection. During pyroptotic cell death, interleukin-1ß (IL-1ß) release occurs through caspase-1 and caspase-11-mediated gasdermin D pore formation. In vivo, responses to lipopolysaccharide (LPS) result in IL-1ß secretion. In vitro, however, murine macrophages require a second "danger signal" for the inflammasome-driven maturation of IL-1ß. Recent reports have shown caspase-8-mediated pyroptosis in LPS-activated macrophages but have provided conflicting evidence regarding the release of IL-1ß under these conditions. Here, to further characterize the mechanism of LPS-induced secretion in vitro, we reveal an important role for cellular FLICE-like inhibitory protein (cFLIP) in the regulation of the inflammatory response. Specifically, we show that deficiency of the long isoform cFLIPL promotes complex II formation, driving pyroptosis, and the secretion of IL-1ß in response to LPS alone.

Constitutive Interferon Attenuates RIPK1/3-Mediated Cytokine Translation.

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are well known for their capacity to drive necroptosis via mixed-lineage kinase-like domain (MLKL). Recently, RIPK1/3 kinase activity has been shown to drive inflammation via activation of MAPK signaling. However, the regulatory mechanisms underlying this kinase-dependent cytokine production remain poorly understood. In the present study, we establish that the kinase activity of RIPK1/3 regulates cytokine translation in mouse and human macrophages. Furthermore, we show that this inflammatory response is downregulated by type I interferon (IFN) signaling, independent of type I IFN-promoted cell death. Specifically, low-level constitutive IFN signaling attenuates RIPK-driven activation of cap-dependent translation initiation pathway components AKT, mTORC1, 4E-BP and eIF4E, while promoting RIPK-dependent cell death. Altogether, these data characterize constitutive IFN signaling as a regulator of RIPK-dependent inflammation and establish cap-dependent translation as a crucial checkpoint in the regulation of cytokine production.

Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis.

Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.

Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses.

Legionella pneumophila elicits caspase-11-driven macrophage pyroptosis through guanylate-binding proteins (GBPs) encoded on chromosome 3. It has been proposed that microbe-driven IFN upregulates GBPs to facilitate pathogen vacuole rupture and bacteriolysis preceding caspase-11 activation. We show here that macrophage death occurred independently of microbial-induced IFN signaling and that GBPs are dispensable for pathogen vacuole rupture. Instead, the host-intrinsic IFN status sustained sufficient GBP expression levels to drive caspase-1 and caspase-11 activation in response to cytosol-exposed bacteria. In addition, endogenous GBP levels were sufficient for the release of DNA from cytosol-exposed bacteria, preceding the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway for Ifnb induction. Mice deficient for chromosome 3 GBPs were unable to mount a rapid IL-1/chemokine (C-X-C motif) ligand 1 (CXCL1) response during Legionella-induced pneumonia, with defective bacterial clearance. Our results show that rapid GBP activity is controlled by host-intrinsic cytokine signaling and that GBP activities precede immune amplification responses, including IFN induction, inflammasome activation, and cell death.