Lieselotte Vande Walle

Non-canonical inflammasome activation targets caspase-11

Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11−/− mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1–/– mice lack both caspase-11 and caspase-1. Interestingly, Casp11–/– macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1–/–Casp11129mt/129mt macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.

Toxic proteins released from mitochondria in cell death

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity. As a consequence, multiple death-promoting factors residing in the mitochondrial intermembrane space are liberated in the cytosol. Pro- and antiapoptotic Bcl-2 family proteins control the release of these mitochondrial proteins by inducing or preventing permeabilization of the outer mitochondrial membrane. Once released into the cytosol, these mitochondrial proteins activate both caspase-dependent and -independent cell death pathways. Cytochrome c was the first protein shown to be released from the mitochondria into the cytosol, where it induces apoptosome formation. Other released mitochondrial proteins include apoptosis-inducing factor (AIF) and endonuclease G, both of which contribute to apoptotic nuclear DNA damage in a caspase-independent way. Other examples are Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease HtrA2/OMI (high-temperature requirement protein A2), which both promote caspase activation and instigate caspase-independent cytotoxicity. The precise mode of action and importance of cytochrome c in apoptosis in mammalian cells has become clear through biochemical, structural and genetic studies. More recently identified factors, for example HtrA2/OMI and Smac/DIABLO, are still being studied intensively in order to delineate their functions in apoptosis. A better understanding of these functions may help to develop new strategies to treat cancer.

Inflammasome-Dependent Release of the Alarmin HMGB1 in Endotoxemia

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

FADD and Caspase-8 Mediate Priming and Activation of the Canonical and Noncanonical Nlrp3 Inflammasomes

The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. In this study, we show that loss of FADD or caspase-8 in a RIP3-deficient background, but not RIP3 deficiency alone, hampered transcriptional priming and posttranslational activation of the canonical and noncanonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli but not the Nlrc4 inflammasome. In addition, FADD deletion prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8–deficient mice had impaired IL-1β production when challenged with LPS or infected with the enteropathogen Citrobacter rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and noncanonical Nlrp3 inflammasome priming and activation.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1–2% of the world’s population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20myel-KO mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20myel-KO mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20myel-KO mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20myel-KO mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

Toll or Interleukin-1 Receptor (TIR) Domain-containing Adaptor Inducing Interferon-β (TRIF)-mediated Caspase-11 Protease Production Integrates Toll-like Receptor 4 (TLR4) Protein- and Nlrp3 Inflammasome-mediated Host Defense against Enteropathogens

Background: C. rodentium and E. coli induce noncanonical Nlrp3 inflammasome activation through caspase-11. Results: TLR4-TRIF are important for caspase-11 expression, caspase-1 activation, and downstream IL-1β and IL-18 production. Conclusion: TLR4-TRIF axis plays an important role in the up-regulation of caspase-11 and activation of noncanonical inflammasome. Significance: Our study identifies novel molecules upstream of caspase-11 that are involved in activation of noncanonical inflammasome. Enteric pathogens represent a major cause of morbidity and mortality worldwide. Toll-like receptor (TLR) and inflammasome signaling are critical for host responses against these pathogens, but how these pathways are integrated remains unclear. Here, we show that TLR4 and the TLR adaptor TRIF are required for inflammasome activation in macrophages infected with the enteric pathogens Escherichia coli and Citrobacter rodentium. In contrast, TLR4 and TRIF were dispensable for Salmonella typhimurium-induced caspase-1 activation. TRIF regulated expression of caspase-11, a caspase-1-related protease that is critical for E. coli- and C. rodentium-induced inflammasome activation, but dispensable for inflammasome activation by S. typhimurium. Thus, TLR4- and TRIF-induced caspase-11 synthesis is critical for noncanonical Nlrp3 inflammasome activation in macrophages infected with enteric pathogens.

Dietary modulation of the microbiome affects autoinflammatory disease

The incidences of chronic inflammatory disorders have increased considerably over the past three decades. Recent shifts in dietary consumption may have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2cmo mice, which express a homozygous Leu98Pro missense mutation in the Pombe Cdc15 homology family protein PSTPIP2 (proline-serine-threonine phosphatase interacting protein 2), spontaneously develop osteomyelitis that resembles chronic recurrent multifocal osteomyelitis in humans. Recent reports demonstrated a crucial role for interleukin-1β (IL-1β) in osteomyelitis, but deletion of the inflammasome components caspase-1 and NLRP3 failed to rescue Pstpip2cmo mice from inflammatory bone disease. Thus, the upstream mechanisms controlling IL-1β production in Pstpip2cmo mice remain to be identified. In addition, the environmental factors driving IL-1β-dependent inflammatory bone erosion are unknown. Here we show that the intestinal microbiota of diseased Pstpip2cmo mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2cmo mice that were fed a diet rich in fat and cholesterol maintained a normal body weight, but were markedly protected against inflammatory bone disease and bone erosion. Diet-induced protection against osteomyelitis was accompanied by marked reductions in intestinal Prevotella levels and significantly reduced pro-IL-1β expression in distant neutrophils. Furthermore, pro-IL-1β expression was also decreased in Pstpip2cmo mice treated with antibiotics, and in wild-type mice that were kept under germ-free conditions. We further demonstrate that combined deletion of caspases 1 and 8 was required for protection against IL-1β-dependent inflammatory bone disease, whereas the deletion of either caspase alone or of elastase or neutrophil proteinase 3 failed to prevent inflammatory disease. Collectively, this work reveals diet-associated changes in the intestinal microbiome as a crucial factor regulating inflammasome- and caspase-8-mediated maturation of IL-1β and osteomyelitis in Pstpip2cmo mice.

Deregulated inflammasome signaling in disease

Summary:  Inflammasomes are multi‐protein complexes that sense microbial molecules and endogenous danger signals in intracellular compartments. Inflammasome assembly results in caspase‐1 activation, which in turn drives maturation and secretion of the pro‐inflammatory cytokines interleukin 1β (IL‐1β) and IL‐18, and induces pyroptosis to eliminate the infectious agent. The importance of inflammasomes in regulating immune responses was recognized with the discovery of polymorphisms in genes encoding inflammasome components and their linkage to aberrant production of IL‐1β and IL‐18 in autoimmune and hereditary periodic fevers syndromes. We review the current knowledge on the role of inflammasomes in regulating innate and adaptive immune responses with an emphasis on the role of these immune complexes in autoinflammatory disorders and autoimmune diseases such as colitis, type I diabetes, multiple sclerosis and vitiligo.

Activation of the NLRP1b inflammasome independently of ASC-mediated caspase-1 autoproteolysis and speck formation

Despite its clinical importance in infection and autoimmunity, the activation mechanisms of the NLRP1b inflammasome remain enigmatic. Here we show that deletion of the inflammasome adaptor ASC in BALB/c mice and in C57BL/6 macrophages expressing a functional NLRP1b prevents anthrax lethal toxin (LeTx)-induced caspase-1 autoproteolysis and speck formation. However, ASC−/− macrophages undergo normal LeTx-induced pyroptosis and secrete significant amounts of interleukin (IL)-1β. In contrast, ASC is critical for caspase-1 autoproteolysis and IL-1β secretion by the NLRC4, NLRP3 and AIM2 inflammasomes. Notably, LeTx-induced inflammasome activation is associated with caspase-1 ubiquitination, which is unaffected in ASC-deficient cells. In vivo, ASC-deficient mice challenged with LeTx produce significant levels of IL-1β, IL-18 and HMGB1 in circulation, although caspase-1 autoproteolysis is abolished. As a result, ASC−/− mice are sensitive to rapid LeTx-induced lethality. Together, these results demonstrate that ASC-driven caspase-1 autoprocessing and speck formation are dispensable for the activation of caspase-1 and the NLRP1b inflammasome.

The inflammasome adaptor ASC regulates the function of adaptive immune cells by controlling Dock2-mediated Rac activation and actin polymerization

The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide–exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes.