Summary Inflammasomes are important sentinels of innate immune defense, sensing pathogens and inducing cell death in infected cells 1 . There are several inflammasome sensors that each detect and respond to specific pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) 1 . In contrast to this one PAMP/DAMP to one sensor specificity, during infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which could contemporaneously engage multiple inflammasome sensors 2 – 5 . Here we discovered that AIM2 regulated the innate immune sensors Pyrin and ZBP1 to drive inflammatory signaling and inflammatory cell death, PANoptosis, and provide host protection during infections with herpes simplex virus 1 (HSV1) and Francisella novicida . We also observed that AIM2, Pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD that drove inflammatory cell death. Collectively, our findings define a previously unknown regulatory connection and molecular interaction among AIM2, Pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that involves multiple inflammasome sensors and cell death regulators. These results represent a new paradigm in understanding the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and Pyrin-mediated diseases.

Cell death provides host defense and maintains homeostasis. Za-containing molecules are essential for these processes. Z-DNA binding protein 1 (ZBP1) activates inflammatory cell death, PANoptosis, whereas adenosine deaminase acting on RNA 1 (ADAR1) serves as an RNA editor to maintain homeostasis. Here, we identify and characterize ADAR1's interaction with ZBP1, defining its role in cell death regulation and tumorigenesis. Combining interferons (IFNs) and nuclear export inhibitors (NEIs) activates ZBP1-dependent PANoptosis. ADAR1 suppresses this PANoptosis by interacting with the Za2 domain of ZBP1 to limit ZBP1 and RIPK3 interactions. Adar1 fl/fl LysM cre mice are resistant to development of colorectal cancer and melanoma, but deletion of the ZBP1 Za2 domain restores tumorigenesis in these mice. In addition, treating wild-type mice with IFN-g and the NEI KPT-330 regresses melanoma in a ZBP1-dependent manner. Our findings suggest that ADAR1 suppresses ZBP1-mediated PANoptosis, promoting tumorigenesis. Defining the functions of ADAR1 and ZBP1 in cell death is fundamental to informing therapeutic strategies for cancer and other diseases.

T cells have a crucial role in viral clearance and vaccine response; however, the mechanisms regulating their responses to viral infections or vaccinations remain elusive. In this study, we investigated T-cell homeostasis, apoptosis, DNA damage, and repair machineries in a large cohort of subjects with hepatitis C virus (HCV) infection. We found that naive CD4 T cells in chronically HCV-infected individuals (HCV T cells) were significantly reduced compared with age-matched healthy subjects. In addition, HCV T cells were prone to apoptosis and DNA damage, as evidenced by increased 8-oxoguanine expression and γH2AX/53BP1-formed DNA damage foci—hallmarks of DNA damage responses. Mechanistically, the activation of DNA repair enzyme ataxia telangiectasia mutated (ATM) was dampened in HCV T cells. ATM activation was also diminished in healthy T cells exposed to ATM inhibitor or to HCV (core protein) that inhibits the phosphoinositide 3 kinase pathway, mimicking the biological effects in HCV T cells. Importantly, ectopic expression of ATM was sufficient to repair the DNA damage, survival deficit, and cell dysfunctions in HCV T cells. Our results demonstrate that insufficient DNA repair enzyme ATM leads to increased DNA damage and renders HCV T cells prone to apoptotic death, which contribute to the loss of naive T cells in HCV infection. Our study reveals a novel mechanism for T-cell dysregulation and viral persistence, providing a new strategy to improve immunotherapy and vaccine responses against human viral diseases.

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.

The myeloid-related protein S100A9 reprograms Gr1CD11b myeloid precursors into myeloid-derived suppressor cells (MDSCs) during murine sepsis. Here, we show that the immunosuppressive cytokine IL-10 supports S100A9 expression and its nuclear localization in MDSCs to function as immune repressors. To support this new concept, we showed that antibody mediated IL-10 blockade in wild-type mice after sepsis induction inhibited MDSC expansion during late sepsis, and that ectopic expression of S100A9 in Gr1CD11b cells from S100A9 knockout mice switched them into the MDSC phenotype only in the presence of IL-10. Knockdown of S100A9 in MDSCs from wild-type mice with late sepsis confirmed our findings in the S100A9 knockout mice. We also found that while both IL-6 and IL-10 can activate S100A9 expression in naive Gr1CD11b cells, only IL-10 can induce S100A9 nuclear localization. These results support that IL-10 drives the molecular path that generates MDSCs and enhances immunosuppression during late sepsis, and inform that targeting this immune repressor path may improve sepsis survival in mice.