Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

Loss of cIAP1 in Endothelial Cells Limits Metastatic Extravasation through Tumor-Derived Lymphotoxin Alpha.

In this study, we determined whether Smac mimetics play a role in metastasis, specifically in circulation, tumor extravasation and growth in a metastatic site. Reports suggest inducing the degradation of IAPs through use of Smac mimetics, alters the ability of the tumor cell to metastasize. However, a role for the immune or stromal compartment in affecting the ability of tumor cells to metastasize upon loss of IAPs has not been defined. To address this open question, we utilized syngeneic tumor models in a late-stage model of metastasis. Loss of cIAP1 in the endothelial compartment, rather than depletion of cIAP2 or absence of cIAP1 in the hematopoietic compartment, caused reduction of tumor load in the lung. Our results underline the involvement of the endothelium in hindering tumor cell extravasation upon loss of cIAP1, in contrast to the immune compartment. Endothelial specific depletion of cIAP1 did not lead to cell death but resulted in an unresponsive endothelium barrier to permeability factors causing a decrease in tumor cell extravasation. Surprisingly, lymphotoxin alpha (LTA), and not TNF, secreted by the tumor cells, was critical for the extravasation. Using TCGA, we found high LTA mRNA expression correlated with decreased survival in kidney carcinoma and associated with advanced disease stage. Our data suggest that Smac mimetics, targeting cIAP1/2, reduce metastasis to the lung by inhibiting tumor cell extravasation.

Correction: TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.

The original version of this article contained an error in the name of one of the co-authors (Erika Owsley). This has been corrected in the PDF and HTML versions.

TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.

The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap-/- macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap-/- myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1ß, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components.