NINJ1 mediates inflammatory cell death, PANoptosis, and drives lethality during heat stress

Innate immunity provides the first line of defense through key mechanisms, including pyrogen and cytokine production and cell death. While elevated body temperature during infection is beneficial, heat stress (HS) can lead to inflammation and pathology. Links between HS, cytokine release, and inflammation have been observed, but fundamental innate immune mechanisms driving pathology during HS remain unclear. Here, we use diverse genetic approaches to elucidate innate immune pathways in HS. Our results show that bacteria and LPS robustly increase inflammatory cell death, PANoptosis, during HS. NINJ1 is the key executioner of this cell death to release inflammatory molecules, independent of other pore-forming executioners. In an in vivo HS model, mortality is reduced by deleting NINJ1 and fully rescued by deleting key PANoptosis molecules. Our findings suggest that therapeutic strategies blocking NINJ1 or its upstream regulators to prevent PANoptosis may reduce the release of inflammatory mediators and benefit patients experiencing HS. Overall design: The Mouse Brie CRISPR KO library carrying four gRNAs for each gene to cover the entire genome was a gift from David Root and John Doench (Addgene #73632 and #73633). The plasmid library was amplified and validated in the Center for Advanced Genome Engineering (CAGE) at St. Jude as described in the Broad GPP protocol, the only exception being the use of Endura DUOs electrocompetent E. coli cells. The St. Jude Hartwell Center Genome Sequencing Facility provided all NGS sequencing. Single end 100 cycle sequencing was performed on a NovaSeq 6000 (Illumina). Validation to check gRNA presence and representation was performed using calc_auc_v1.1.py (https://github.com/mhegde/) and count_spacers.py84. Viral particles were produced by the St. Jude Vector Development and Production laboratory using co-transfection of the lentiviral packaging plasmids along with the Brie library in the 293T cells. CRISPR KO screens were analyzed using Mageck-Vispr v0.5.7. Cas9-expressing iBMDMs were generated from Cas9-GFP knock-in mice. A total of 300 × 10^6 Cas9-iBMDMs were distributed across twelve 15 cm2 tissue culture dishes at 25 × 10^6 cells per dish and infected with the Brie library of lentiviral particles, which carry four gRNAs for each gene to cover the entire genome, at an MOI of 0.3 in 25 ml of complete DMEM (DMEM supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; S1620, Biowest)) with 100 ml of LentiBOOST transduction reagent (SB-P-LV-101-02, Lentivirus Transduction Enhancer Solution, Siron Biotech). After infection, the iBMDMs were incubated for 24 h for ef?cient transduction. These transduced cells were expanded with intermittent passaging to avoid overcrowding of the cells and to generate a suf?cient number of cells for the downstream whole-genome CRISPR screens. Two replicates of an adequate number of cells were used as control to obtain a representation (screen depth) of > 500 cells for each sgRNA of the library, and a similar number of cells from the same batch of virus preparation were stimulated with LPS priming for 2 h, then incubated at 43 °C for 1 h to induce heat shock (HS). After 24 h, the media was removed, and cells in the unstimulated and stimulated conditions were washed with PBS to remove the non-adherent dead cells, leaving only the adherent surviving cells for downstream analyses. The surviving cells from the unstimulated and LPS plus HS-treated samples were then subjected to CRISPR screen enrichment analysis. Total genomic DNA was isolated using NucleoSpin® Blood kits (Takara Bio Inc., USA; 740954 and 740950) and the concentrations of the isolated gDNA samples were measured using NanoDrop (Thermo Fisher Scientific, USA). Next-generation sequencing (NGS) of the PCR-amplified, barcoded gRNAs was performed to quantitatively identify gRNAs that were enriched in the surviving pool of cells. Because the presence of a gRNA should delete the corresponding gene in that cell, enriched gRNAs were expected to represent genes where deletion rescues the cells from LPS plus HS-mediated cell death, suggesting they are positive regulators of the cell death..