Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter

Abstract

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Fig. 1. Challenge with H. polygyrus and S. mansoni eggs reactivates MHV68

(A) C57BL6/J mice were infected intraperitoneally (i.p.) with MHV68-M3-FL and challenged with H. polygyrus 42 days later. Mice were imaged prior to H. polygyrus infection (day 0) and 5, 7 and 9 days after. Three representative mice imaged on days 0 and 7 are shown. (B) Total flux (photons/second) was quantitated for mice in 2 independent experiments for the timecourse after infection with H. polygyrus. Data from 4 independent experiments at day 7 post H. polygyrus is also shown. (C) C57BL/6J mice were infected intranasally (i.n.) with MHV68-M3-FL. Diagram indicates timecourse of experiment and challenge with Sm eggs or PBS as a control. Mice were injected with D-Luciferin and imaged prior to intravenous (i.v.) challenge with Sm eggs (day 0). They were subsequently imaged 5, 8 and 11 days after challenge with Sm eggs. Three representative mice imaged on days 0 and 8 are shown. (D) Total flux (photons/second) was quantitated from mice in two independent experiments after Sm egg challenge. Symbols represent individual mice, and the mean and standard error are indicated * p<0.05, ** p<0.01, *** p<0.001 by 2-way repeated measures ANOVA with Tukey’s and Bonferroni’s post-test.

Fig. 2. IL-4 and IFNγ signatures identified in different macrophage populations during MHV68 infection

(A) GSEA analysis of virus-positive and virus-negative cells sorted from the peritoneum of MHV68-infected mice compared to BMDMs stimulated with IL-4 or IFNγ/LPS. (B) C57BL/6J mice or YARG/R26-stop-RFP mice were either mock infected or infected with MHV68-cre. CD11b+F4/80+ cells were gated and examined for RFP and YFP expression. Representative plots from two independent experiments, with three to five mice per experiment. (C) Quantitation of FACS analysis in (B) with each symbol representing a single mouse.

Fig. 3. IL-4 promotes viral replication and antagonizes IFNγ suppression of viral replication through direct binding to a viral promoter

(A) BMDMs were untreated or treated with the indicated doses of IL-4 and infected with MHV68. 24 hours post infection cells were analyzed for expression of MHV68 lytic viral proteins and CD206 expression. Represents three independent experiments. (B) Pretreatment of BMDMs with IL-4, IL-13, or IL-5 prior to infection with MHV68 and FACS analysis. (C) BMDMs were pretreated with varying doses of IFNγ ± 10 ng/ml of IL-4. 24 hours post infection cells were analyzed for expression of lytic viral proteins. Represents three independent experiments. (D) Gene 50 expression was analyzed by RT-PCR in BMDMs pretreated with IL-4 and/or IFNγ. Expression was normalized to Gapdh. Represents four independent experiments. (E) RAW264.7 cells were transfected with vectors expressing luciferase under control of four different gene 50 promoters ((23, 24). Cells were then treated ± IL-4 or IL-13 for 24 hours, lysed and assayed for luciferase activity. (F) Cells were transfected with the N4/N5 promoter luciferase construct and treated with IL-4, IFNγ, or both. (G) N4/N5 luciferase mutants were transfected into RAW264.7 cells and assayed for sensitivity to IL-4. (H) RAW264.7 cells were infected with MHV68 at MOI=5 and treated with IL-4 for 8 hours. After chromatin immunoprecipitation with Stat6 antibody, quantitative PCR was performed for the N4/N5 promoter region or VEGF. Percent of input after normalizing to IgG control was calculated for both N4/N5 and VEGF. 1 experiment representative of 4 independent experiments is shown. (I) Schematic of N4/N5 luciferase construct with potential Stat-binding mutants. n.d. not detected. n.s. not significant. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by T test or 1-way ANOVA with Sidak’s multiple comparisons test.

Fig. 4. MHV68 and KSHV reactivation from latency is regulated by IL-4

(A) C57BL6/J mice were infected with MHV68-M3-FL i.p. 42 days later mice were imaged for luciferase expression (d0) and then received isotype control (PIP), anti-IFNγ (H22), IL4c, or both anti-IFNγ and IL-4c. On day 44 mice received a second dose of IL4c or PBS. Mice were imaged five days after the first treatment and total flux from the abdominal region was quantitated. Four representative mice are shown. (B) Quantitation of total flux from three individual experiments described in (A) are shown. Each symbol represents an individual mouse. Bars are means, and error bars are standard errors. (C) WT or Stat6KO mice were treated as in (A) with anti-IFNγ/IL4c and imaged 5 days later. Bars are the means of individual mice (symbols), and error bars are standard error. (D) Experimental set-up was the same as Fig. 1A. Latently infected WT and Stat6KO mice infected with MHV68-M3-FL were infected with H. polygyrus or treated with PBS on day 42 and reactivation was quantitated by luciferase induction 7 days later. (E) BCBL-1 cells were treated with IL-4 for 3 or 5 days or TPA for 48 hours and viral gene expression was analyzed. Shown is the fold increase in gene expression over mock after normalization of GAPDH. Data from 3 independent experiments. (F) Supernatants from cells treated in (E) were collected and virus was isolated by centrifugation. Viral genome copy number was assayed by qPCR using serial diluted LANA expression plasmid as a standard curve. 1 experiment representative of 2 independent experiments is shown. For luciferase experiments: * p<0.05, ** p<0.01, n.s. not significant by 2-way repeated measures ANOVA with Tukey’s and Bonferroni’s post-test.