LXR Alpha Restricts Gammaherpesvirus Reactivation from Latently Infected Peritoneal Cells

Abstract

Gammaherpesviruses are ubiquitous viruses that establish lifelong infections. Importantly, these viruses are associated with numerous cancers and lymphoproliferative diseases. While risk factors for developing gammaherpesvirus-driven cancers are poorly understood, it is clear that elevated viral reactivation from latency often precedes oncogenesis. Here, we demonstrate that the liver X receptor alpha isoform (LXRα) restricts gammaherpesvirus reactivation in an anatomic-site-specific manner. We have previously demonstrated that deficiency of both LXR isoforms (α and β) leads to an increase in fatty acid and cholesterol synthesis in primary macrophage cultures, with a corresponding increase in gammaherpesvirus replication. Interestingly, expression of fatty acid synthesis genes was not derepressed in LXRα-deficient hosts, indicating that the antiviral effects of LXRα are independent of lipogenesis. Additionally, the critical host defenses against gammaherpesvirus reactivation, virus-specific CD8⁺ T cells and interferon (IFN) signaling, remained intact in the absence of LXRα. Remarkably, using a murine gammaherpesvirus 68 (MHV68) reporter virus, we discovered that LXRα expression dictates the cellular tropism of MHV68 in the peritoneal cavity. Specifically, LXRα^-/- mice exhibit reduced latency within the peritoneal B cell compartment and elevated latency within F4/80⁺ cells. Thus, LXRα restricts gammaherpesvirus reactivation through a novel mechanism that is independent of the known CD8⁺ T cell-based antiviral responses or changes in lipid synthesis and likely involves changes in the tropism of MHV68 in the peritoneal cavity.IMPORTANCE Liver X receptors (LXRs) are nuclear receptors that mediate cholesterol and fatty acid homeostasis. Importantly, as ligand-activated transcription factors, LXRs represent potential targets for the treatment of hypercholesterolemia and atherosclerosis. Here, we demonstrate that LXRα, one of the two LXR isoforms, restricts reactivation of latent gammaherpesvirus from peritoneal cells. As gammaherpesviruses are ubiquitous oncogenic agents, LXRs may represent a targetable host factor for the treatment of poorly controlled gammaherpesvirus infection and associated lymphomagenesis.

Keywords: chronic infection; gammaherpesvirus; liver X receptor alpha; peritoneal B cells.

FIG 1

LXRα restricts MHV68 reactivation from peritoneal cells, but not splenocytes, in chronically infected animals. Mice of the indicated genotypes were intraperitoneally infected with 500 PFU wild-type MHV68. Mice were euthanized at 16 (A and B), 28 (C and D), or 42 (E and F) days postinfection, and splenocytes (right) or peritoneal exudate (left) cells were processed into single-cell suspensions. The cells were serially diluted and cocultured with MEFs to assess the frequency of cells reactivating latent virus (A, C, and E) or subjected to nested-PCR analysis to assess the frequency of cells harboring viral genomes (B, D, and F). The data are pooled from 2 to 4 independent experiments. The error bars represent standard errors of the mean. CPE, cytopathic effect. (G) The percentages of peritoneal cells positive for MHV68 DNA or supporting ex vivo reactivation were calculated for each time point using average frequencies of pooled data presented in panels A to F. Reactivation efficiency was defined as a ratio of percent reactivating to percent viral DNA-positive cells for each time point.

FIG 2

LXRα deficiency does not result in increased expression of proviral lipogenic genes during chronic MHV68 infection. Mice of the indicated genotypes were intraperitoneally infected with 500 PFU wild-type MHV68 for 28 days or mock infected. Peritoneal cells were isolated and subjected to qRT-PCR analysis to assess expression of the indicated genes. Gapdh, glyceraldehyde-3-phosphate dehydrogenase. Data are pooled from two independent experiments. Each symbol represents an individual animal, means and standard errors of the mean are shown. *, P < 0.05; **, P < 0.01; ****, P < .0001.

FIG 3

MHV68-specific T cell responses remain intact in LXRα^−/− mice. Mice of the indicated genotypes were intraperitoneally infected with 500 PFU wild-type MHV68 or mock infected. At 28 days postinfection, the mice were euthanized and splenocytes and peritoneal cells were processed into single-cell suspensions. Cells were stained with α-CD3, α-CD8, and α-CD44 antibodies, and MHC class I tetramers were loaded with the immunodominant peptides from the viral ORF6 (B, C, F, and G) or ORF61 (D, E, H, and I) proteins. (A) Representative flow plots from stained peritoneal cells. (B to I) Frequencies and total numbers of virus-specific CD8⁺ T cells for peritoneal cells (B to E) and splenocytes (F to I). The data are pooled from two independent experiments. Each symbol represents an individual animal; means and standard errors of the mean are shown.

FIG 4

LXRα does not regulate expression of IFN-γ by MHV68-specific CD8⁺ T cells. Mice of the indicated genotypes were intraperitoneally infected with 500 PFU wild-type MHV68 or mock infected. At 28 days postinfection, the mice were euthanized, and peritoneal cells were isolated. The peritoneal cells were stimulated with 10 μg/ml ORF6_487–495 and ORF61_524–531 immunodominant peptides in the presence of 10 μg/ml brefeldin A for 6 h at 37°C. Cells were stained for flow cytometric analysis with α-CD3 and α-CD8 and intracellularly with α-IFN-γ antibodies. (A) Representative flow plots illustrating IFN-γ production. (B to E) Summarized frequencies and total numbers of IFN-γ-producing CD8⁺ T cells from the peritoneal cavity (B and C) and spleen (D and E). (F) MFI of IFN-γ signal from IFN-γ⁺ peritoneal CD8⁺ T cells. The data are pooled from at least two independent experiments. Each symbol represents an individual animal; means and standard errors of the mean are shown.

FIG 5

LXRα expression promotes neither signaling nor antiviral activity of IFN-γ. (A and B) Bone marrow-derived macrophages were treated with 10 U/ml IFN-β, 1 U/ml IFN-γ, or vehicle control for 30 min (A) or 7 h (B). (A) Total and phosphorylated STAT1 proteins were measured by Western blotting in two biological repeats for each condition. (B) MNDA expression was measured by qRT-PCR. The results of two independent experiments were pooled, and the symbols represent biological replicates. (C) Peritoneal cells isolated from mice of the indicated genotypes at 28 days postinfection were subjected to limiting-dilution analyses to assess the frequency of viral reactivation. Cultures were treated with 5 U/ml IFN-γ or vehicle control (Veh) immediately following plating to assess the ability of exogenous IFN-γ to suppress MHV68 reactivation. The data were pooled from two independent experiments. (D) Serum was collected before infection and at 14 and 28 days postinfection. IFN-γ concentrations were determined by ELISA. The data are pooled from at least two independent experiments, and each symbol represents an individual animal. (E and F) Mice of the indicated genotypes were intraperitoneally infected with MHV68 as for Fig. 1 or mock infected. At 28 days postinfection, RNA was isolated from peritoneal cells immediately following harvest. Each symbol represents an individual animal. The data are representative of the results of two independent experiments (A) or pooled from two or more independent experiments (B to F). Error bars represent the standard error of the mean.

FIG 6

LXRα expression dictates latent viral tropism or gene expression in peritoneal cells. Mice of the indicated genotypes were intraperitoneally infected with MHV68.ORF73βla for 28 days. Splenocytes and peritoneal cells were isolated and stained for CD19 and F4/80 surface markers. The cells were then loaded with the fluorescent β-lactamase substrate CCF2-AM for 1 h at room temperature. (A and B) Bulk splenocytes (A) and peritoneal cells (B) were examined for mLANA expression via flow cytometry, as indicated by the fluorescent signal from cleaved CCF2. (C) mLANA⁻ and mLANA⁺ cells were gated and examined for CD19 and F4/80 expression. Data from two independent experiments are summarized.