Cleansing the cytosol: Most human cells, not just those belonging to the immune system, mount protective responses to infection when activated by the immune cytokine interferon-gamma (IFN-γ). How IFN-γ confers this function in nonimmune cells and tissues is poorly understood. Gaudet et al. used genome-scale CRISPR/Cas9 gene editing to identify apolipoprotein L-3 (APOL3) as an IFN-γ–induced bactericidal protein that protects human epithelium, endothelium, and fibroblasts against infection (see the Perspective by Nathan). APOL3 directly targets bacteria in the host cell cytosol and kills them by dissolving their anionic membranes into lipoprotein complexes. This work reveals a detergent-like mechanism enlisted during human cell-autonomous immunity to combat intracellular pathogens. Science , abf8113, this issue p. eabf8113 ; see also abj5637, p. 276

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo–electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our “open conformer” model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.

The immunologic consequences of using bactericidal versus bacteriostatic antibiotic treatments are unclear. We observed a bacteriostatic (growth halting) treatment was more protective than a bactericidal (bacteria killing) treatment in a murine peritonitis model. To understand this unexpected difference, we compared macrophage responses to bactericidal treated bacteria or bacteriostatic treated bacteria. We found that Gram-negative bacteria treated with bactericidal drugs induced more proinflammatory cytokines than those treated with bacteriostatic agents. Bacterial DNA – released only by bactericidal treatments – exacerbated inflammatory signaling through TLR9. Without TLR9 signaling, the in vivo efficacy of bactericidal drug treatment was rescued. This demonstrates that antibiotics can act in important ways distinct from bacterial inhibition: like causing treatment failure by releasing DNA that induces excessive inflammation. These data establish a novel link between how an antibiotic affects bacterial physiology and subsequent immune system engagement, which may be relevant for optimizing treatments to simultaneously clear bacteria and modulate inflammation.

The enzyme NDPK-D promotes changes in mitochondrial function that support inflammasome activation.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.

Precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells and electrochemistry at the electrode-electrolyte interface. Here, we introduce the use of microfabricated electrodes based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), which substantially increases cell viability and transfection efficiency. As a proof of concept, we demonstrate the enhanced delivery of Cas9 protein, guide RNA, and plasmid DNA into cell lines and primary cells. This use of PEDOT:PSS enables rapid modification of difficult-to-transfect cell types to accelerate their study and use as therapeutic platforms.