Ethanol-HIV Stimulates Macrophage-derived Extracellular Vesicles to Promote a Profibrotic Phenotype in Hepatic Stellate Cells

Liver fibrosis is the scarring process where excessive extracellular matrix proteins occur and can be caused by exposure to certain toxins or compounds such as alcohol. Alcohol can lead to increased fibrosis and cirrhosis in people living with HIV due to its ability to influence the liver’s microenvironment. Extracellular vesicles (EVs) communicate between cells by transferring their cargo. Under stress, macrophages can communicate with hepatic cells by releasing EVs and potentially progressing liver disease. The current study examines how ethanol affects EVs production from HIV-infected macrophages and how macrophage-derived EVs modulate profibrotic phenotype in hepatic stellate cells. Monocyte-derived macrophages (MDM) were infected with HIV and then exposed to 50 mM EtOH during incubation. The THP-1 monocytes were differentiated to macrophages with PMA (5 ng/mL) before alcohol and HIV treatment. The medium from the macrophages was collected for ultracentrifugation to isolate the EVs. The EVs were quantified using Nanoparticle tracking analysis (NTA). Transcriptional expression of genes was performed with qPCR. LX-2 hepatic stellate cells were exposed to macrophage-derived EVs from different treatment groups to assess profibrotic activation. Ethanol treatment in HIV-infected macrophages increased the production of EVs compared to their respective controls. The majority of the EVs from the MDM cells were in the range of small EVs (50-200 nm). Exposure of EtOH-HIV-induced macrophage EVs to LX2 cells significantly increased the transcriptional expression of profibrotic genes Col1A1, ACTA2, and CTGF. Combined treatment of EtOH and HIV in macrophages downregulated the hsa-miR92a-3p expression in macrophage-derived EVs that binds with its putative target Col1A1 to increase fibrotic changes in recipient LX-2 cells. The findings of this study lead to the conclusion that a combination of ethanol and HIV stimulates macrophage derived EVs with the downregulation of miR92a, which will activate the profibrotic phenotype in hepatic stellate cells. This activation will contribute to the progression of liver disease.https://digitalcommons.unmc.edu/surp2021/1022/thumbnail.jp

Neuronal-Derived Extracellular Vesicles are Enriched in the Brain and Serum of HIV-1 Transgenic Rats

Despite the efficacy of combination antiretroviral therapy (ART) in controlling human immunodeficiency virus (HIV-1) replication, cytotoxic viral proteins such as HIV-1 transactivator of transcription (Tat) persist in tissues such as the brain. Although HIV-1 does not infect neuronal cells, it is susceptible to viral Tat protein-mediated toxicity, leading to neuroinflammation that underlies HIV-associated neurocognitive disorders (HAND). Given the role of extracellular vesicles (EVs) in both cellular homoeostasis and under pathological conditions, we sought to investigate the alterations in the quantity of neuronal-derived EVs in the brain–as defined by the presence of cell adhesion molecule L1 (L1CAM) and to evaluate the presence of L1CAM+ EVs in the peripheral circulation of HIV-1 transgenic (HIV-1 Tg) rats. The primary goal of this study was to investigate the effect of long-term exposure of HIV-1 viral proteins on the release of neuronal EVs in the brain and their transfer in the systemic compartment. Brain and serum EVs were isolated from both wild type and HIV-1 Tg rats using differential ultracentrifugation with further purification using the Optiprep gradient method. The subpopulation of neuronal EVs was further enriched using immunoprecipitation. The current findings demonstrated increased presence of L1CAM+ neuronal-derived EVs both in the brain and serum of HIV-1 Tg rats. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles

Human-like NSG Mouse Glycoproteins Sialylation Pattern Changes the Phenotype of Human Lymphocytes and Sensitivity to HIV-1 Infection

BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins\u27 chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγ CONCLUSION: NSG-cma

Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection

Abstract Background The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins’ chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. Results We mutated mouse CMAH in the NOD/scid-IL2Rγc −/− (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8. Conclusion NSG-cmah −/− mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins