Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation.

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

Tyrosine kinase inhibitors can activate the NLRP3 inflammasome in myeloid cells through lysosomal damage and cell lysis.

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)-including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)-activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow-derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

Inflammasomes activate the protease caspase-1, which cleaves interleukin-1ß and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1C284A, we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1C284A, we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis.

The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis.

BACKGROUND: Standardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for Escherichia coli, such tools are still missing for the Gram-positive model organism Bacillus subtilis. The goal of this study was to develop and thoroughly evaluate such a genetic toolbox. RESULTS: We developed five BioBrick-compatible integrative B. subtilis vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the lacZ and luxABCDE cassettes, were BioBrick-adjusted, to enable ß-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the Bacillus BioBrick Box also provides five widely used epitope tags (FLAG, His10, cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice. CONCLUSION: Our genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with B. subtilis. We believe that our well-documented and carefully evaluated Bacillus BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other BioBrick-based project in B. subtilis.