**TNF Family Cytokines Induce Distinct Cell Death Modalities in the A549 Human Lung Epithelial Cell Line when Administered in Combination with Ricin Toxin**

#### **Alexa L. Hodges 1,**†**, Cody G. Kempen 1,**†**, William D. McCaig 1, Cory A. Parker 1, Nicholas J. Mantis 2,\* and Timothy J. LaRocca 1,\***


Received: 19 June 2019; Accepted: 28 July 2019; Published: 1 August 2019

**Abstract:** Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and is classified as a biothreat agen<sup>t</sup> by the Centers for Disease Control and Prevention (CDC). Inhalation, the most potent route of toxicity, triggers an acute respiratory distress-like syndrome that coincides with near complete destruction of the lung epithelium. We previously demonstrated that the TNF-related apoptosis-inducing ligand (TRAIL; CD253) sensitizes human lung epithelial cells to ricin-induced death. Here, we report that ricin/TRAIL-mediated cell death occurs via apoptosis and involves caspases -3, -7, -8, and -9, but not caspase-6. In addition, we show that two other TNF family members, TNF-α and Fas ligand (FasL), also sensitize human lung epithelial cells to ricin-induced death. While ricin/TNF-<sup>α</sup>- and ricin/FasL-mediated killing of A549 cells was inhibited by the pan-caspase inhibitor, zVAD-fmk, evidence suggests that these pathways were not caspase-dependent apoptosis. We also ruled out necroptosis and pyroptosis. Rather, the combination of ricin plus TNF-α or FasL induced cathepsin-dependent cell death, as evidenced by the use of several pharmacologic inhibitors. We postulate that the effects of zVAD-fmk were due to the molecule's known off-target effects on cathepsin activity. This work demonstrates that ricin-induced lung epithelial cell killing occurs by distinct cell death pathways dependent on the presence of different sensitizing cytokines, TRAIL, TNF-<sup>α</sup>, or FasL.

**Keywords:** ricin; toxins; cytokines; toxin-mediated diseases; apoptosis; cathepsin; tumor necrosis factor; fas; caspases

**Key Contribution:** In this study, we have defined the cell death pathways induced by ricin/TRAIL, ricin/TNF-<sup>α</sup>, and ricin/FasL in human lung epithelial cells. As our analysis probed each pathway at several steps, this work may lead to novel therapeutic approaches to ricin toxicity that target multiple cell death pathways at different steps in addition to direct targeting of ricin.
