establishment-dual-humanized-tk-nog-mouse-model-for-hiv-associated

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

University of Nebraska Medical Center

Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4 cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection  could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis.This article has an associated First Person interview with the first author of the paper.

Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice.

HIV-HCV co-infection causes rapid progression of liver fibrosis. These outcomes to liver cirrhosis can be improved, but not stopped by specific antiviral therapies. Due to high significance of HIV-HCV interactions for morbidity and mortality in co-infected patients, our attention was attracted to the multi-component pathogenesis of fibrosis progression as the transition to end-stage liver disease development. In this study, we hypothesize that increased matrix stiffness enhances apoptosis in HCV-HIV-co-infected hepatocytes and that capturing of apoptotic bodies (AB) derived from these infected hepatocytes by hepatic stellate cells (HSC) drives the fibrosis progression. As the source of viruses, JFH1 (HCV genotype 2a) and HIV-1 (either purified or containing in infected macrophage supernatants) were chosen. Using Huh7.5-CYP (RLW) cells and primary human hepatocytes mono-infected with HCV and HIV or co-infected, we have shown that both HCV and HIV RNA levels were increased in co-infected cells, which was accompanied by hepatocyte apoptosis. This apoptosis was attenuated by azidothymidine treatment. The levels of both infections and apoptosis were more prominent in primary hepatocytes cultured on substrates mimicking fibrotic stiffness (24 kPa-stiff) compared to substrates mimicking healthy liver (2.4 kPa-soft). The engulfment of AB from pathogen-exposed hepatocytes activated pro-fibrotic mRNAs in HSC. Overall, the increased matrix stiffness is not only a consequence of liver inflammation/fibrosis, but the condition that further accelerates liver fibrosis development. This is attributed to the switching of HSC to pro-fibrotic phenotype by capturing of excessive amounts of apoptotic HCV- and HIV-infected hepatocytes.

Matrix stiffness regulate apoptotic cell death in HIV-HCV co-infected hepatocytes: Importance for liver fibrosis progression.

Alcohol-induced progression of hepatitis C virus (HCV) infection is related to dysfunction of innate immunity in hepatocytes. Endogenously produced interferon (IFN)α induces activation of interferon-stimulated genes (ISGs) via triggering of the Janus kinase-signal transducer and activator of transcription 1 (STAT1) pathway. This activation requires protein methyltransferase 1-regulated arginine methylation of STAT1. Here, we aimed to study whether STAT1 methylation also depended on the levels of demethylase jumonji domain-containing 6 protein (JMJD6) and whether ethanol and HCV affect JMJD6 expression in hepatocytes.

Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism.

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.

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

Long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs), play an important regulatory role in controlling various biological processes. Both in vitro and in vivo studies have demonstrated that lincRNA-Cox2 plays a global regulatory role in regulating the expression of immune genes. Extracellular vesicles (EVs) are cell-derived nanosized membrane vesicles that have gained increasing attention in recent years due to their ability to efficiently deliver therapeutics to specific target organs or cell types. In this study, we found that lincRNA-Cox2 controls the expression of a set of cell cycle genes in lipopolysaccharide (LPS)-stimulated microglial cells. Our in vitro study suggested that knocking down lincRNA-Cox2 reversed LPS-induced microglial proliferation. In addition, our in vivo study demonstrated that intranasally delivered lincRNA-Cox2-siRNA loaded EVs could reach the brain resulting in a significant decrease in the expression of lincRNA-Cox2 in the microglia. Importantly, lincRNA-Cox2-siRNA loaded EVs also decreased LPS-induced microglial proliferation in mice. These findings indicate that intranasal delivery of EV-loaded small RNA could be developed as therapeutics for treatment of a multitude of CNS disorders.

Intranasal Delivery of lincRNA-Cox2 siRNA Loaded Extracellular Vesicles Decreases Lipopolysaccharide-Induced Microglial Proliferation in Mice.

Extracellular vesicles (EVs) are nanosized, membrane-bound vesicles released from eukaryotic and prokaryotic cells that can transport cargo containing DNA, RNA, lipids and proteins, between cells as a means of intercellular communication. Although EVs were initially considered to be cellular debris deprived of any essential biological functions, emerging literature highlights the critical roles of EVs in the context of intercellular signaling, maintenance of tissue homeostasis, modulation of immune responses, inflammation, cancer progression, angiogenesis, and coagulation under both physiological and pathological states. Based on the ability of EVs to shuttle proteins, lipids, carbohydrates, mRNAs, long non-coding RNAs (lncRNAs), microRNAs, chromosomal DNA, and mitochondrial DNA into target cells, the presence and content of EVs in biofluids have been exploited for biomarker research in the context of diagnosis, prognosis and treatment strategies. Additionally, owing to the characteristics of EVs such as stability in circulation, biocompatibility as well as low immunogenicity and toxicity, these vesicles have become attractive systems for the delivery of therapeutics. More recently, EVs are increasingly being exploited as conduits for delivery of therapeutics for anticancer strategies, immunomodulation, targeted drug delivery, tissue regeneration, and vaccination. In this review, we highlight and discuss the multiple strategies that are employed for the use of EVs as delivery vehicles for therapeutic agents, including the potential advantages and challenges involved. Graphical abstract.

Article	Year
Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice. Biology open\| PubMed ID: 29361613	2018
Matrix stiffness regulate apoptotic cell death in HIV-HCV co-infected hepatocytes: Importance for liver fibrosis progression. Biochemical and biophysical research communications\| PubMed ID: 29679566	2018
Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 29693039	2018
Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection. BMC immunology\| PubMed ID: 30616506	2019
Intranasal Delivery of lincRNA-Cox2 siRNA Loaded Extracellular Vesicles Decreases Lipopolysaccharide-Induced Microglial Proliferation in Mice. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology\| PubMed ID: 31325121	2019
Strategies for the use of Extracellular Vesicles for the Delivery of Therapeutics. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology\| PubMed ID: 31456107	2019

Raghubendra Singh Dagur

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