Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1ß release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.

A1 is induced by pathogen ligands to limit myeloid cell death and NLRP3 inflammasome activation.

Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro-survival BCL-2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post-translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK-dependent apoptosis and the subsequent NLRP3 inflammasome-dependent and inflammasome-independent maturation of the inflammatory cytokine IL-1ß. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL-1ß activation triggered by Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs). Consequently, A1-deficient mice exhibit heightened IL-1ß production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host-directed antimicrobial therapeutics.

CCNE1 and survival of patients with tubo-ovarian high-grade serous carcinoma: An Ovarian Tumor Tissue Analysis consortium study.

BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis Consortium Study.

BACKGROUND: Recently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC. METHODS: Two prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis. RESULTS: Consistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67-0.91, p < 0.0001). GMNN protein expression was not significantly associated with overall HSGC patient survival. However, HGSCs with >35% GMNN expression showed a trend for better outcomes, though this was not significant. CONCLUSION: We provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

Evaluation of a health and social care programme to improve outcomes following critical illness: a multicentre study.

RATIONALE: At present, clinicians aiming to support patients through the challenges after critical care have limited evidence to base interventions. OBJECTIVES: Evaluate a multicentre integrated health and social care intervention for critical care survivors. A process evaluation assessed factors influencing the programme implementation. METHODS: This study evaluated the impact of the Intensive Care Syndrome: Promoting Independence and Return to Employment (InS:PIRE) programme. We compared patients who attended this programme with a usual care cohort from the same time period across nine hospital sites in Scotland. The primary outcome was health-related quality of life (HRQoL) measured via the EuroQol 5-dimension 5-level instrument, at 12 months post hospital discharge. Secondary outcome measures included self-efficacy, depression, anxiety and pain. RESULTS: 137 patients who received the InS:PIRE intervention completed outcome measures at 12 months. In the usual care cohort, 115 patients completed the measures. The two cohorts had similar baseline demographics. After adjustment, there was a significant absolute increase in HRQoL in the intervention cohort in relation to the usual care cohort (0.12, 95% CI 0.04 to 0.20, p=0.01). Patients in the InS:PIRE cohort also reported self-efficacy scores that were 7.7% higher (2.32 points higher, 95% CI 0.32 to 4.31, p=0.02), fewer symptoms of depression (OR 0.38, 95% CI 0.19 to 0.76, p=0.01) and similar symptoms of anxiety (OR 0.58, 95% CI 0.30 to 1.13, p=0.11). There was no significant difference in overall pain experience. Key facilitators for implementation were: integration with inpatient care, organisational engagement, flexibility to service inclusion; key barriers were: funding, staff availability and venue availability. CONCLUSIONS: This multicentre evaluation of a health and social care programme designed for survivors of critical illness appears to show benefit at 12 months following hospital discharge.

Profiling the immune landscape in mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients. METHODS: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration. RESULTS: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates. CONCLUSION: In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies.

No longer married to inflammasome signaling: the diverse interacting pathways leading to pyroptotic cell death.

For over 15 years the lytic cell death termed pyroptosis was defined by its dependency on the inflammatory caspase, caspase-1, which, upon pathogen sensing, is activated by innate immune cytoplasmic protein complexes known as inflammasomes. However, this definition of pyroptosis changed when the pore-forming protein gasdermin D (GSDMD) was identified as the caspase-1 (and caspase-11) substrate required to mediate pyroptotic cell death. Consequently, pyroptosis has been redefined as a gasdermin-dependent cell death. Studies now show that, upon liberation of the N-terminal domain, five gasdermin family members, GSDMA, GSDMB, GSDMC, GSDMD and GSDME can all form plasma membrane pores to induce pyroptosis. Here, we review recent research into the diverse stimuli and cell death signaling pathways involved in the activation of gasdermins; death and toll-like receptor triggered caspase-8 activation of GSDMD or GSMDC, apoptotic caspase-3 activation of GSDME, perforin-granzyme A activation of GSDMB, and bacterial protease activation of GSDMA. We highlight findings that have begun to unravel the physiological situations and disease states that result from gasdermin signaling downstream of inflammasome activation, death receptor and mitochondrial apoptosis, and necroptosis. This new era in cell death research therefore holds significant promise in identifying how distinct, yet often networked, pyroptotic cell death pathways might be manipulated for therapeutic benefit to treat a range of malignant conditions associated with inflammation, infection and cancer.

CXCL11 expressing C57BL/6 mice have intact adaptive immune responses to viral infection.

The chemokine receptor CXCR3 is expressed on immune cells to co-ordinate lymphocyte activation and migration. CXCR3 binds three chemokine ligands, CXCL9, CXCL10 and CXCL11. These ligands display distinct expression patterns and ligand signaling biases; however, how each ligand functions individually and collaboratively is incompletely understood. CXCL9 and CXCL10 are considered pro-inflammatory chemokines during viral infection, while CXCL11 may induce a tolerizing state. The investigation of the individual role of CXCL11 in vivo has been hampered as C57BL/6 mice carry several mutations that result in a null allele. Here, CRISPR/Cas9 was used to correct these mutations on a C57BL/6 background. It was validated that CXCL11KI mice expressed CXCL11 protein in dendritic cells, spleen and lung. CXCL11KI mice were largely phenotypically indistinguishable from C57BL/6 mice, both at steady-state and during two models of viral infection. While CXCL11 expression did not modify acute antiviral responses, this study provides a new tool to understand the role of CXCL11 in other experimental settings.

FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins.

Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.

p53 and ovarian carcinoma survival: an Ovarian Tumor Tissue Analysis consortium study.

Our objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi-institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36-3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11-2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC.

The escape of Candida albicans from macrophages is enabled by the fungal toxin candidalysin and two host cell death pathways.

The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay reveals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes: the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macrophages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.

Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death.

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing.

Necroptotic movers and shakers: cell types, inflammatory drivers and diseases.

The necroptotic cell death pathway has received significant attention for its ability to trigger inflammatory responses and its potential involvement in related conditions. Recent insights into the essential membrane damaging necroptotic pseudokinase effector, Mixed lineage kinase domain like (MLKL), have revealed a number of diverse MLKL functions that contribute to the inflammatory nature of necroptosis. Here we review distinct MLKL signalling roles and document the immunogenic molecules released by necroptosis. We discuss specific in vivo MLKL-driven responses, the activation of inflammasome complexes and innate lymphoid cells, which have been documented to drive disease. Finally, we list necroptotic competent cell types and their involvement in MLKL-driven cell death-associated and inflammatory-associated conditions.