Mechanistic basis for potassium efflux-driven activation of the human NLRP1 inflammasome.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.

Visualization of accessible cholesterol using a GRAM domain-based biosensor.

Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated.

Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis.

The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate immunity by activating the human NLRP1 inflammasome. Here, we report that ZAKα and NLRP1 sense bacterial exotoxins that target ribosome elongation factors. One such toxin, diphtheria toxin (DT), the causative agent for human diphtheria, triggers RSR-dependent inflammasome activation in primary human keratinocytes. This process requires iron-mediated DT production in the bacteria, as well as diphthamide synthesis and ZAKα/p38-driven NLRP1 phosphorylation in host cells. NLRP1 deletion abrogates IL-1ß and IL-18 secretion by DT-intoxicated keratinocytes, while ZAKα deletion or inhibition additionally limits both pyroptotic and inflammasome-independent non-pyroptotic cell death. Consequently, pharmacologic inhibition of ZAKα is more effective than caspase-1 inhibition at protecting the epidermal barrier in a 3D skin model of cutaneous diphtheria. In summary, these findings implicate ZAKα-driven RSR and the NLRP1 inflammasome in antibacterial immunity and might explain certain aspects of diphtheria pathogenesis.

Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection.

Bat cells and tissue have elevated basal expression levels of antiviral genes commonly associated with interferon alpha (IFNα) signaling. Here, we show Interferon Regulatory Factor 1 (IRF1), 3, and 7 levels are elevated in most bat tissues and that, basally, IRFs contribute to the expression of type I IFN ligands and high expression of interferon regulated genes (IRGs). CRISPR knockout (KO) of IRF 1/3/7 in cells reveals distinct subsets of genes affected by each IRF in an IFN-ligand signaling-dependent and largely independent manner. As the master regulators of innate immunity, the IRFs control the kinetics and maintenance of the IRG response and play essential roles in response to influenza A virus (IAV), herpes simplex virus 1 (HSV-1), Melaka virus/Pteropine orthoreovirus 3 Melaka (PRV3M), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) infection. With its differential expression in bats compared to that in humans, this highlights a critical role for basal IRF expression in viral responses and potentially immune cell development in bats with relevance for IRF function in human biology.

Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia.

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.