Nadia Luheshi

Deubiquitinases Regulate the Activity of Caspase-1 and Interleukin-1β Secretion via Assembly of the Inflammasome

Background: The inflammasome is a multimolecular complex that regulates the processing of the pro-inflammatory cytokine interleukin-1β. Results: Inhibitors of deubiquitinase (DUB) enzymes inhibited the release of interleukin-1β. Conclusion: DUBs regulate assembly of the inflammasome. Significance: DUBs may represent new anti-inflammatory drug targets for the treatment of inflammatory disease. IL-1β is a potent pro-inflammatory cytokine produced in response to infection or injury. It is synthesized as an inactive precursor that is activated by the protease caspase-1 within a cytosolic molecular complex called the inflammasome. Assembly of this complex is triggered by a range of structurally diverse damage or pathogen associated stimuli, and the signaling pathways through which these act are poorly understood. Ubiquitination is a post-translational modification essential for maintaining cellular homeostasis. It can be reversed by deubiquitinase enzymes (DUBs) that remove ubiquitin moieties from the protein thus modifying its fate. DUBs present specificity toward different ubiquitin chain topologies and are crucial for recycling ubiquitin molecules before protein degradation as well as regulating key cellular processes such as protein trafficking, gene transcription, and signaling. We report here that small molecule inhibitors of DUB activity inhibit inflammasome activation. Inhibition of DUBs blocked the processing and release of IL-1β in both mouse and human macrophages. DUB activity was necessary for inflammasome association as DUB inhibition also impaired ASC oligomerization and caspase-1 activation without directly blocking caspase-1 activity. These data reveal the requirement for DUB activity in a key reaction of the innate immune response and highlight the therapeutic potential of DUB inhibitors for chronic auto-inflammatory diseases.

Interleukin-1α expression precedes IL-1β after ischemic brain injury and is localised to areas of focal neuronal loss and penumbral tissues

BackgroundCerebral ischemia is a devastating condition in which the outcome is heavily influenced by inflammatory processes, which can augment primary injury caused by reduced blood supply. The cytokines interleukin-1α (IL-1α) and IL-1β are key contributors to ischemic brain injury. However, there is very little evidence that IL-1 expression occurs at the protein level early enough (within hours) to influence brain damage after stroke. In order to determine this we investigated the temporal and spatial profiles of IL-1α and IL-1β expression after cerebral ischemia.FindingsWe report here that in mice, as early as 4 h after reperfusion following ischemia induced by occlusion of the middle cerebral artery, IL-1α, but not IL-1β, is expressed by microglia-like cells in the ischemic hemisphere, which parallels an upregulation of IL-1α mRNA. 24 h after ischemia IL-1α expression is closely associated with areas of focal blood brain barrier breakdown and neuronal death, mostly near the penumbra surrounding the infarct. The sub-cellular distribution of IL-1α in injured areas is not uniform suggesting that it is regulated.ConclusionsThe early expression of IL-1α in areas of focal neuronal injury suggests that it is the major form of IL-1 contributing to inflammation early after cerebral ischemia. This adds to the growing body of evidence that IL-1α is a key mediator of the sterile inflammatory response.

Dual functionality of interleukin-1 family cytokines: implications for anti-interleukin-1 therapy.

Dysregulated inflammation contributes to disease pathogenesis in both the periphery and the brain. Cytokines are coordinators of inflammation and were originally defined as secreted mediators, released from expressing cells to activate plasma membrane receptors on responsive cells. However, a group of cytokines is now recognized as having dual functionality. In addition to their extracellular effects, these cytokines act inside the nuclei of cytokine‐expressing or cytokine‐responsive cells. Interleukin‐1 (IL‐1) family cytokines are key pro‐inflammatory mediators, and blockade of the IL‐1 system in inflammatory diseases is an attractive therapeutic goal. All current therapies target IL‐1 extracellular actions. Here we review evidence that suggests IL‐1 family members have dual functionality. Several IL‐1 family members have been detected inside the nuclei of IL‐1‐expressing or IL‐1‐responsive cells, and intranuclear IL‐1 is reported to regulate gene transcription and mRNA splicing. However, further work is required to determine the impact of IL‐1 intranuclear actions on disease pathogenesis. The intranuclear actions of IL‐1 family members represent a new and potentially important area of IL‐1 biology and may have implications for the future development of anti‐IL‐1 therapies.

Sphingosine regulates the NLRP3‐inflammasome and IL‐1β release from macrophages

Interleukin‐1β (IL‐1β) is a pro‐inflammatory cytokine that regulates inflammatory responses to injury and infection. IL‐1β secretion requires the protease caspase‐1, which is activated following recruitment to inflammasomes. Endogenous danger‐associated molecular patterns (DAMPs) released from necrotic cells activate caspase‐1 through an NLRP3‐inflammasome. Here, we show that the endogenous lipid metabolite sphingosine (Sph) acts as a DAMP by inducing the NLRP3‐inflammasome‐dependent secretion of IL‐1β from macrophages. This process was dependent upon serine/threonine protein phosphatases since the PP1/PP2A inhibitors okadaic acid and calyculin A inhibited Sph‐induced IL‐1β release. IL‐1β release induced by other well‐characterized NLRP3‐inflammasome activators, such as ATP and uric acid crystals, in addition to NLRC4 and AIM2 inflammasome activators was also blocked by these inhibitors. Thus, we propose Sph as a new DAMP, and that a serine/threonine phosphatase (PP1/PP2A)‐dependent signal is central to the endogenous host mechanism through which diverse stimuli regulate inflammasome activation.

Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

Sterile inflammation is a host response to tissue injury that is mediated by damage‐associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro‐inflammatory cytokine IL‐1α is reported to be a damage‐associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL‐1α is produced by microglia, the resident brain macrophages. We found that IL‐1α is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL‐1α to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen‐glucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL‐1α released significantly less IL‐1α than microglia with predominantly cytosolic IL‐1α. The remaining IL‐1α was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL‐1α may serve to limit inflammation following cell death.

Zinc depletion regulates the processing and secretion of IL-1β.

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.

The Dynamics and Mechanisms of Interleukin‐1α and β Nuclear Import

Pro‐inflammatory members of the interleukin‐1 (IL‐1) family of cytokines (IL‐1α and β) are important mediators of host defense responses to infection but can also exacerbate the damaging inflammation that contributes to major human diseases. IL‐1α and β are produced by cells of the innate immune system, such as macrophages, and act largely after their secretion by binding to the type I IL‐1 receptor on responsive cells. There is evidence that IL‐1α is also a nuclear protein that can act intracellularly. In this study, we report that both IL‐1α and IL‐1β produced by microglia (central nervous system macrophages) in response to an inflammatory challenge are distributed between the cytosol and the nucleus. Using IL‐1‐β‐galactosidase and IL‐1‐green fluorescent protein chimeras (analyzed by fluorescence recovery after photobleaching), we demonstrate that nuclear import of IL‐1α is exclusively active, requiring a nuclear localization sequence and Ran, while IL‐1β nuclear import is entirely passive. These data provide valuable insights into the dynamic regulation of intracellular cytokine trafficking.

Transformation of the tumour microenvironment by a CD40 agonist antibody correlates with improved responses to PD-L1 blockade in a mouse orthotopic pancreatic tumour model.

Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.

Boron-Based Inhibitors of the NLRP3 Inflammasome

Summary NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimers disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.

Th1 cytokines are more effective than Th2 cytokines at licensing anti-tumour functions in CD40-activated human macrophages in vitro

CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T‐cell‐activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T‐cell‐derived cytokines license anti‐tumour functions in CD40‐activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL‐10, in a similar fashion to bacterial LPS, but failed to promote anti‐tumour functions. The Th1 cytokine IFN‐γ optimally licensed CD40L‐induced macrophage anti‐tumour functions, inducing a switch from IL‐10 to IL‐12p70 production, promoting macrophage‐mediated Th1 T‐cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL‐4 and IL‐13 promoted IL‐12p70 production (albeit without inhibiting IL‐10 production) and enhanced Th1 T‐cell skewing by CD40L‐activated macrophages. However, IL‐4 and IL‐13 did not enhance tumouricidal activity in CD40L‐activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L‐activated macrophages. The presence of tumour‐infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.