Persistent Replication of HIV, Hepatitis C Virus (HCV), and HBV Results in Distinct Gene Expression Profiles by Human NK Cells

Abstract

Natural killer (NK) cells during chronic viral infection have been well studied in the past. We performed an unbiased next-generation RNA-sequencing approach to identify commonalities or differences of the effect of HIV, HCV, and HBV viremia on NK cell transcriptomes. Using cell sorting, we obtained CD3^- CD56⁺ NK cells from blood of 6 HIV-, 8 HCV-, and 32 HBV-infected patients without treatment. After library preparation and sequencing, we used an in-house analytic pipeline to compare expression levels with matched healthy controls. In NK cells from HIV-, HCV-, and HBV-infected patients, transcriptome analysis identified 272, 53, and 56 differentially expressed genes, respectively (fold change, >1.5; q-value, 0.2). Interferon-stimulated genes were induced in NK cells from HIV/HCV patients, but not during HBV infection. HIV viremia downregulated ribosome assembly genes in NK cells. In HBV-infected patients, viral load and alanine aminotransferase (ALT) variation had little effect on genes related to NK effector function. In conclusion, we compare, for the first time, NK cell transcripts of viremic HIV, HCV, and HBV patients. We clearly demonstrate distinctive NK cell gene signatures in three different populations, suggestive for a different degree of functional alterations of the NK cell compartment compared to healthy individuals.IMPORTANCE Three viruses exist that can result in persistently high viral loads in immunocompetent humans: human immunodeficiency virus (HIV), hepatitis C virus, and hepatitis B virus. In the last decades, by using flow cytometry and in vitro assays on NK cells from patients with these types of infections, several impairments have been established, particularly during and possibly contributing to HIV viremia. However, the background of NK cell impairments in viremic patients is not well understood. In this study, we describe the NK cell transcriptomes of patients with high viral loads of different etiologies. We clearly demonstrate distinctive NK cell gene signatures with regard to interferon-stimulated gene induction and the expression of genes coding for activation markers or proteins involved in cytotoxic action, as well immunological genes. This study provides important details necessary to uncover the origin of functional and phenotypical differences between viremic patients and healthy subjects and provides many leads that can be confirmed using future in vitro manipulation experiments.

Keywords: hepatitis B virus; hepatitis C virus; human immunodeficiency virus; natural killer cells.

FIG 1

NK cells of HIV-infected patients differentially express numerous genes encoding NK activating surface markers and cytotoxicity-related genes and display a downregulation of ribosome assembly genes. (A) Representative flow cytometry plots showing the gating strategy for peripheral CD56⁺ CD3⁻ NK cells used for all included subjects. (B) Summary of the post-sort purity percentages of CD56⁺ CD3⁻ cells in virally infected and healthy subjects. (C) Representative flow cytometry plots showing comparable pre- and post-sort percentages in healthy individuals in comparison with HIV-, HCV-, and HBV-infected patients. (D) Relative expression of the 10 most upregulated genes in HIV-, HCV-, and HBV-infected individuals compared to matched healthy controls (fold change, ≥1.5; q-value, 0.2), including several common ISGs in HIV/HCV infection. NK cells derived from HIV-infected individuals strikingly downregulated several genes related to ribosome biogenesis (RPL27, RPS7, RPL24, RPS13, and RPL10L), and during both HIV and HCV infection, ISGs (IFI6 and IFI27L2) were among the top 10 most upregulated genes.

FIG 2

NK cell ISG are not induced by HBV, but both HIV and HCV infection have parallel patterns of upregulated ISGs. (A) Expression relative to matched healthy subjects of several ISGs induced during chronic HCV and HIV infections that is indicative of parallel ISG induction in NK cells. Chronic infection with HCV and HIV induces the expression of several ISGs, but in HBV patients only a single NK cell ISG was upregulated compared to healthy controls. (B) Relative NK cell ISG expression of NK cells from HIV, HBV, and HCV patients and healthy subjects. Each row represents a single ISG. No large clusters of up- or downregulation are common to the viremic subjects, confirming that the expression patterns of individual subjects vary considerably. Patients are presented in the following order: healthy control and HBV, HCV, and HIV infections. The colors represent a range of fold differences from −1.5 (blue) to 1.5 (red).

FIG 3

Variation of serum HBV DNA and ALT levels among four HBV cohorts is associated with variation of NK cell immunological genes but has minimal impact on the expression of individual genes related to cytotoxicity or IFN signaling. (A) Graphical representation of the patient characteristics of the HBV-infected cohort. Based on serum HBV DNA, ALT levels, and HBeAg presence at the time of sampling, patients were categorized into four clinical HBV phases according to international guidelines (European Association for the Study of the Liver, 2017). The HBsAg levels are presented to illustrate lower values among IC and ENEG patients which are characteristic for the natural history of HBV. (B) Total number of DEGs in each HBV clinical phase. The numbers of upregulated (rightward) or downregulated (leftward) genes per phase are shown as horizontal bars. (C) The top up- or downregulated genes are depicted per phase compared to healthy individuals. Regardless of serum ALT levels, no ISGs were induced, except for IRF4. IRF4 was highly expressed in all phases, except for the ENEG phase. Among the genes upregulated in the IA phase, a few are immune related, including FCRL2/5, PDLIM2, and SIT1.