Tumour necrosis factor-α induces macromolecule translocation in HIVderived duodenal organoids

Kopano Valerie Masete ^1* Alain S. Massarani ¹ Jörg-Dieter Schulzke ¹ Hans-Jörg Epple ^2,3 Nina A. Hering ^4*

• ¹ Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology and Rheumatology, Campus Benjamin Franklin Charité - Universitätsmedizin Berlin, Berlin, Germany
• ² Department of Gastroenterology, Rheumatology and Infectious Diseases, Campus Benjamin Franklin Charité - Universitätsmedizin Berlin, Berlin, Germany
• ³ Antibiotic Stewardship Team, Medical Directorate, Campus Benjamin Franklin Charité - Universitätsmedizin Berlin, Berlin, Baden-Württemberg, Germany
• ⁴ Department of General, Visceral and Vascular Surgery, Charité University Medicine Berlin, Berlin, Germany

Background: Disease progression from human immunodeficiency virus (HIV) infection to acquired immunodeficiency syndrome (AIDS) is marked by chronic immune activation, partly due to increased translocation of gut-derived microbial antigens. Elevated mucosal tumour necrosis factor-α (TNF-α) and resulting epithelial cell apoptosis may be the etiology. However, studies using carcinoma cell lines have failed to find a causal link, possibly due to cellular abnormalities related to the malignant transformation of these immortal cell lines. Methods: We established intestinal organoid monolayers from healthy controls and HIV-infected adults and characterized their growth dynamics and cellular composition. We then examined the effects of HIV-associated cytokines on transepithelial resistance (TER), apoptosis and macromolecule translocation. Results: Organoid monolayers from HIV-infected patients grew similarly to healthy controls, forming confluent monolayers within one to two weeks containing enterocytes, Paneth, goblet and stem cells. IFN-γ synergized with TNF-α, allowing TNF-α to cause caspase-mediated apoptosis and TER reduction within 5 ± 3 hours, reflecting patient sample heterogeneity. This led to paracellular transport of 4 kDa Dextran and transcytosis of 44 kDa horse radish peroxidase, both of which could be blocked by pan-caspase inhibitor, Q-VD-Oph. Conclusion: Our study confirms that intestinal organoid monolayers from biopsies of HIV-infected individuals can be used to study apoptosis-related epithelial barrier dysfunction and macromolecular translocation. We provide direct evidence that TNF-α-induced apoptosis triggered two pathways of macromolecular translocation: paracellular transport via apoptotic leaks and transcytosis. Therapies targeting apoptosis may be useful in preventing disease progression from HIV to AIDS.