George R. Dubyak

Interleukin 1-beta (IL-1β) is an inflammatory cytokine produced by myeloid cells in response to infection or sterile tissue damage. Secretion of bioactive IL-1β from macrophages (Mφ) or dendritic cells (DC) downstream of activated NLRP3/caspase-1 inflammasomes is the best characterized model; this is mediated by caspase-1 cleavage of proIL-1β and Gasdermin D. Gasdermin D pores that form in the plasma membrane mediate IL-1β release and pyroptotic cell death. NLRP3 inflammasome assembly is triggered by perturbation of ionic, metabolic or organelle homeostasis via diverse stimuli. A recent report demonstrated that NLRP3 activators in Mφ/DC include tyrosine kinase inhibitors such as imatinib mesylate used as frontline chemotherapeutics for chronic myelogenous leukemia (CML). This action of imatinib was initiated by lysosomal membrane permeabilization (LMP). As CML is characterized by high numbers of circulating immature granulocytes and neutrophils, we assessed the effects of imatinib on NLRP3 inflammasome signaling in murine and human neutrophils. We report that imatinib-treated neutrophils can process and release IL-1β independently of NLRP3 inflammasome assembly and the expression/activity of caspase-1 or Gasdermin D. Mechanistically, imatinib induces azurophilic granule permeabilization to drive robust cytosolic accumulation of granule-derived neutral serine proteases, serine protease-mediated processing of proIL-1β, and release of mature IL-1β. Together these findings elucidate a novel mechanism by which disruption of neutrophil granules can bypass the NLRP3 inflammasome pathway to drive serine protease-mediated IL-1β processing and release.

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis, with symptoms including pruritus, pancreatitis, fat malabsorption, intestinal inflammation, and failure to thrive. High-throughput studies showed interconnection between ATP8B1 and phosphoinositide (PIPs), but the mechanism linking ATP8B1, lipid metabolism, and inflammation remains unclear. Atp8b1 ^G308V/G308V mouse model, unbiased RNAseq, high-resolution-stimulation emission depltion (STED)-microscopy, and Crispr-Cas9 generated ATP8B1 ^−/− knockouts in hepatocytes/monocytes/macrophages were used to determine role of ATP8B1 in phosphatidylinositol,4-5-bisphosphate (PIP2) trafficking and inflammation. Human ATP8B1, purified from Sf9 insect cells and reconstituted in proteoliposomes, was used to test cell-free PIP2 flip. Various in-vitro techniques were used for testing direct interaction between PIP2 and ATP8B1. ATP8B1 maintains PIP2 at the inner leaflet of plasma membrane (PM). ATP8B1 flips PIP2 in cells, without altering flip of PE or bulk-endocytosis. ATP8b1 flips PIP2 in a cell-free system. ATP8B1 deletion promotes bile-salt-mediated cholesterol extraction from hepatocytes in a PIP2-dependent manner. PIP2 directly binds to the P-loop of ATP8B1. Unbiased RNAseq showed upregulation of inflammatory cytokines in ATP8b1 ^−/− immune cells. ATP8B1 ^−/− monocytes/macrophages showed aberrant lipopolysaccharide (LPS)-induced cleavage of GSDMD, formation of GSDMD pores, and interleukin-1beta (IL1β) release. Inflammation-resolving efferocytosis was impaired in ATP8B1 ^−/− macrophages. Biophysical properties of PM were altered in ATP8b1 ^−/− cells, with the mechanism being disrupted localization of PIP2. Atp8b1 ^G308V/G308V mice exposed to LPS showed higher plasma IL1β and lower survival rates vs. WT mice. ATP8B1 maintains PIP2 at the inner leaflet of PM. ATP8b1 directly flips and binds PIP2. ATP8B1 regulates LPS-induced GsdmD cleavage, formation of GsdmD pores, IL1β release, and mortality in mice.

Steroid resistance poses a major challenge for the management of autoimmune neuroinflammation. T helper 17 (T _H 17) cells are widely implicated in the pathology of steroid resistance; however, the underlying mechanisms are unknown. In this study, we identified that interleukin-1 receptor (IL-1R) blockade rendered experimental autoimmune encephalomyelitis (EAE) mice sensitive to dexamethasone (Dex) treatment. Interleukin-1β (IL-1β) induced a signal transducer and activator of transcription 5 (STAT5)–mediated steroid-resistant transcriptional program in T _H 17 cells, which promoted inflammatory cytokine production and suppressed Dex-induced anti-inflammatory genes. T _H 17-specific deletion of STAT5 ablated the IL-1β–induced steroid-resistant transcriptional program and rendered EAE mice sensitive to Dex treatment. IL-1β synergized with Dex to promote the STAT5-dependent expression of CD69 and the development of central nervous system (CNS)–resident CD69 ⁺ T _H 17 cells. Combined IL-1R blockade and Dex treatment ablated CNS-resident T _H 17 cells, reduced EAE severity, and prevented relapse. CD69 ⁺ tissue-resident T _H 17 cells were also detected in brain lesions of patients with multiple sclerosis. These findings (i) demonstrate that IL-1β–STAT5 signaling in T _H 17 cells mediates steroid resistance and (ii) identify a therapeutic strategy for reversing steroid resistance in T _H 17-mediated CNS autoimmunity.

Neutrophil extracellular traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and cause tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibited the growth of PIK3CA-mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors were associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of ROS in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, entered CRC cells through the RAGE cell surface protein. The internalized CTSG cleaved 14-3-3 proteins, released BAX, and triggered apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.

Summary: NINJ1 is a recently identified active executioner of plasma membrane rupture (PMR), a process previously thought to be a passive osmotic lysis event in lytic cell death. NINJ2 is a close paralog of NINJ1 but it failed to mediate PMR. By cryoEM, we found that both NINJ1 and NINJ2 were able to assemble into linear filament that binds strongly to lipids on one side but is water-soluble on the other side. The more-or-less straight NINJ1 filament was able to wrap around a membrane bleb and solubilize it from the plasma membrane to induce PMR; however, the intrinsically curved NINJ2 filament failed to do so, explaining its incapability of mediating PMR. We further demonstrated that binding to cholesterol at the inner leaflet of the lipid bilayer was responsible for the curving of the NINJ2 filament, while strong lipid binding at the outer leaflet was contributing to NINJ1’s capability of mediating PMR.

Summary: Pyroptosis is a proinflammatory mode of lytic cell death mediated by accumulation of plasma membrane (PM) macropores composed of gasdermin‐family (GSDM) proteins. It facilitates two major functions in innate immunity: (i) elimination of intracellular replicative niches for pathogenic bacteria; and (ii) non‐classical secretion of IL‐1 family cytokines that amplify host‐beneficial inflammatory responses to microbial infection or tissue damage. Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL‐1β release during inflammasome signaling have been extensively characterized in macrophages. This involves cleavage of GSDMD by caspase‐1 to generate GSDMD macropores that mediate IL‐1β efflux and progression to pyroptotic lysis. Neutrophils, which rapidly accumulate in large numbers at sites of tissue infection or damage, become the predominant local source of IL‐1β in coordination with their potent microbiocidal capacity. Similar to macrophages, neutrophils express GSDMD and utilize the same spectrum of diverse inflammasome platforms for caspase‐1‐mediated cleavage of GSDMD. Distinct from macrophages, neutrophils possess a remarkable capacity to resist progression to GSDMD‐dependent pyroptotic lysis to preserve their viability for efficient microbial killing while maintaining GSDMD‐dependent mechanisms for export of bioactive IL‐1β. Rather, neutrophils employ cell‐specific mechanisms to conditionally engage GSDMD‐mediated pyroptosis in response to bacterial pathogens that use neutrophils as replicative niches. GSDMD and pyroptosis have also been mechanistically linked to induction of NETosis, a signature neutrophil pathway that expels decondensed nuclear DNA into extracellular compartments for immobilization and killing of microbial pathogens. This review summarizes a rapidly growing number of recent studies that have produced new insights, unexpected mechanistic nuances, and some controversies regarding the regulation of, and roles for, neutrophil inflammasomes, pyroptosis, and GSDMs in diverse innate immune responses.

Pyroptosis is a mechanism of programmed, necrotic cell death mediated by gasdermins, a family of pore-forming proteins. Caspase-1 activates gasdermin D (GSDMD) under inflammatory conditions, whereas caspase-3 activates GSDME under apoptotic conditions, such as those induced by chemotherapy. These pathways are thought to be separate. However, we found that they are part of an integrated network of gatekeepers that enables pyroptotic cell death. We observed that GSDMD was the primary pyroptotic mediator in cultured blood cells in response to doxorubicin and etoposide, two common chemotherapies for hematopoietic malignancies. Upon treatment, the channel protein pannexin-1 (PANX1), which is stimulated by the initiation of apoptosis, increased membrane permeability to induce K ⁺ efflux-driven activation of the NLRP3 inflammasome and GSDMD. However, either PANX1 or GSDME could also be the primary mediator of chemotherapy-induced pyroptosis when present at higher amounts. The most abundant pore-forming protein in acute myeloid leukemias from patients predicted the cell death pathway in response to chemotherapy. This interconnected network, a multistep switch that converts apoptosis to pyroptosis, could be clinically titratated to modulate cell death with regard to antitumor immunity or tumor lysis syndrome in patients.

The C-type lectin receptor Mincle is known for its important role in innate immune cells in recognizing pathogen and damage associated molecular patterns. Here we report a T cell–intrinsic role for Mincle in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Genomic deletion of Mincle in T cells impairs TH17, but not TH1 cell-mediated EAE, in alignment with significantly higher expression of Mincle in TH17 cells than in TH1 cells. Mechanistically, dying cells release β-glucosylceramide during inflammation, which serves as natural ligand for Mincle. Ligand engagement induces activation of the ASC-NLRP3 inflammasome, which leads to Caspase8-dependent IL-1β production and consequentially TH17 cell proliferation via an autocrine regulatory loop. Chemical inhibition of β-glucosylceramide synthesis greatly reduces inflammatory CD4+ T cells in the central nervous system and inhibits EAE progression in mice. Taken together, this study indicates that sensing of danger signals by Mincle on TH17 cells plays a critical role in promoting CNS inflammation.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1β. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1β–containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro–IL-1β, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1β colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63⁺ sEVs containing IL-1β, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1β–containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1β sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.