HIV-induced mitochondrial ROS and cell death in hepatic stellate cells contribute to a profibrotic profile

CEVALLOS CINTIA; JARMOLUK PATRICIO; SVIERCZ FRANCO; LOPEZ ALICIA; FREIBERGER NICOLE; GUANO ALEX; DELPINO M VICTORIA; QUARLERI JORGE

Congreso; IAS 2023, the 12th IAS Conference on HIV Science; 2023

Background: Hepatic stellate cells (HSC) are susceptible to HIV infection. Whether HIV is able to activate HSCs by damage-related stimuli to secrete excessive extracellular matrix, leading to collagen deposition, is undefined. Since the level of reactive oxygen species (ROS) increases sharply in activated HSCs and HIV exposure may trigger ROS early after viral exposure, our goal is to define the HIV role in propitiating a profibrotic profile by inducing ROS imbalance and cell death among HSC.Methods: Both cell-free X4-tropic HIV (pNL43-GFP 100ng/μL p24 antigen) and HIV-infected T lymphocytes (HIV-GFP+ Jurkat cells labeled with violet proliferation dye, 5:1 ratio) were used to examine the LX-2´s permissiveness to HIV replication. Additionally, LX-2 were subjected to conditioned media (supernatant from HIV-infected T lymphocytes). Viral replication and HIV infection effectiveness were measured 3 days after exposure (dpe) using HIV-p24 antigen by ELISA in supernatants and GFP+ cells by flow cytometry, respectively. In LX-2, cell-death (PCD, Annexin-V/7AAD) and mitochondrial ROS (MitoSox™) levels were measured using flow cytometry, using staurosporine (1μΜ) and rotenone (10μM) as positive controls, respectively. Collagen deposition in the extracellular matrix was determined by Sirius Red staining and spectrophotometric (O.D. 550nm) quantification. All experiments were carried out in triplicate.Results: At 3 dpe, neither the cell-free virus nor the cell-to-cell contact challenge produced HIV progeny. Cell-to-cell contact with HIV-infected T-lymphocytes significantly enhanced necrosis (7AAD+ only) in LX-2 compared to control (1.4% vs. 2.0%; p 0.01). However, no significant differences were observed early (Annexin-V+ only) and late apoptosis (Annexin-V+/7AAD+) (3.6% vs 3.2% and 2.4% vs 3.8% p>0.05) respectively. This was also accompanied by a very early (2 hpe) increase in mitochondrial ROS generation (1.3% vs. 2.3%, p 0.01). Additionally, the extracellular matrix of these cells revealed a 1.6x change in collagen deposition (p = 0.01). When LX-2 were exposed to conditioned media, these anomalies were not observed.Conclusion: Although HSCs are susceptible but not permissive to HIV-X4 tropic infection, their cell-to-cell contact with HIV-infected T lymphocytes results in oxidative stress and enhanced cell death that favors an exacerbated deposition of extracellular matrix, ultimately resulting in a profibrotic profile.