Helminthic larval stage induces cellular apoptosis via caspase 9-mediated mitochondrial dysfunction

Leonardo Elias Sternkopf^1,2Ulrich Fabien Prodjinotho^1,2*Vitka Gres³Nikolaus Repgen⁴Katja Steiger⁵Julia Schluckebier^1,2Chummy S. Sikasunge⁶Dominik Stelzle²Charles Makasi^7,8Andrea Sylvia Winkler^10,2,9Bernard James Ngowi^11,8Nelly Villalobos¹²Friederike Ebner⁴Georg Häcker¹³Philipp Henneke^14,3Clarissa Prazeres da Costa^1,15,2*

• ¹Institute for Medical Microbiology, Immunology and Hygiene, TUM School of Medicine, Technical University of Munich (TUM), Munich, Bavaria, Germany
• ²Center for Global Health, TUM School of Medicine, Technical University of Munich (TUM), Munich, Bavaria, Germany
• ³Institute for Infection Prevention and Control and Center for Chronic Immunodeficiency (CCI), Medical Center and Faculty of Medicine, University of Freiburg, Freiburg, Germany
• ⁴Infection Pathogenesis, School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
• ⁵Institute of Pathology, Faculty of Medicine, Technical University of Munich, Munich, Bavaria, Germany
• ⁶Department of Paraclinical Studies, School of Veterinary Medicine, University of Zambia, Lusaka, Lusaka, Zambia
• ⁷Kilimanjaro Christian Medical University College, Moshi, Kilimanjaro, Tanzania
• ⁸National Institute for Medical Research, Muhimbili Research Centre, Dar es Salaam, Tanzania
• ⁹Department of Neurology, TUM School of Medicine, Technical University of Munich (TUM), Munich, Germany, Munich, Germany
• ¹⁰Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway
• ¹¹Mbeya College of Health and Allied Sciences, University of Dar es Salaam, Dar es Salaam, Dar es Salaam, Tanzania
• ¹²Departamento de Patología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico
• ¹³Institute of Medical Microbiology and Hygiene, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
• ¹⁴Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany
• ¹⁵German Center for Infection and Research (DZIF), Partner Site Munich, Munich 81675, Munich, Bavaria, Germany

Introduction: In human neurocysticercosis (NCC), the cellular and molecular mechanisms of host-parasite interactions triggering brain inflammation and epileptic seizures in Sub-Saharan Africa are poorly understood and associated with the viability of the cyst of the pork tapeworm Taenia solium. We have previously shown that while viable cyst-released molecules promote immune regulation and often asymptomatic disease, the fluid from degenerating cysts causes inflammation in microglia and peripheral immune cells, potentially driving immune-mediated pathology. This study aims to elucidate the apoptotic signaling pathways underlying this process and their relevance for symptomatic disease in NCC patients. Materials and methods: Human and porcine peripheral and brain cells were exposed to T. solium cyst vesicular fluid (CVF). Apoptosis signaling pathways were analysed using flow cytometric FLICA caspase 8 and 9 assays, while mitochondrial dysfunction was assessed via TMRE and MitoTracker Deep Red and Green fluorescent probes. Apoptosis-inducing molecules were identified by differential mass spectrometry and functionally tested using specific inhibitors. Caspase activity and soluble mediators (FasL, ROS, TNFα) were measured in NCC asymptomatic and symptomatic patients' sera, and inflammatory cell infiltrates expressing caspases near viable/degenerating cysts in infected pig brain slices were examined. Results: We found that CVF primarily induced apoptosis and caspase 3 and 9 activity, and minimal necrosis, in a dose-dependent manner across central and peripheral cells. This effect was prominent in CD16+ monocytes, microglia, and in T cell-expressing caspase 3 near degenerating brain cysts. Apoptotic signaling was predominantly mediated by a dynamic remodeling of caspase 9 pathway, accompanied by a loss of mitochondrial potential and a sharp decrease in Bid and Bcl2 transcription, favoring the intrinsic over the FasL-dependent extrinsic pathway and mechanisms. This process is primarily mediated by small molecules (< 30 kDa), and remained unaffected by heat and proteinase treatment. Notably, symptomatic NCC patients exhibited elevated FasL levels correlating with increased caspase activity, underscoring the potential contribution of apoptosis to disease pathogenesis. Conclusions: This study identifies caspase 9-mediated apoptosis as a mechanism of helminth-induced brain inflammation and implicates FasL in symptomatic disease progression. These insights enhance our understanding of NCC immunopathogenesis and may inform future therapeutic strategies targeting apoptotic pathways.