IL-1β/TNF-α/IL-6 inflammatory cytokines promote STAT1-dependent induction of CH25H in Zika virus-infected human macrophages

Abstract

Zika virus (ZIKV)³ is an enveloped, single-stranded, positive-sense RNA virus of the Flaviviridae family that has emerged as a public health threat because of its global transmission and link to microcephaly. Currently there is no vaccine for this virus. Conversion of cholesterol to 25-hydroxycholesterol by cholesterol 25-hydroxylase (CH25H) has been shown to have broad antiviral properties. However, the molecular basis of induction of CH25H in humans is not known. Elucidation of signaling and transcriptional events for induction of CH25H expression is critical for designing therapeutic antiviral agents. In this study, we show that CH25H is induced by ZIKV infection or Toll-like receptor stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines, including IL-1β, tumor necrosis factor α, and IL-6, and this induction depends on the STAT1 transcription factor. Additionally, we observed that cAMP-dependent transcription factor (ATF3) weakly binds to the CH25H promoter, suggesting cooperation with STAT1. However, ZIKV-induced CH25H was independent of type I interferon. These findings provide important information for understanding how the Zika virus induces innate inflammatory responses and promotes the expression of anti-viral CH25H protein.

Keywords: cholesterol 25-hydroxylase; flavivirus; infection; inflammation; innate immunity; macrophage.

Figure 1.

Expression of CH25H with ZIKV exposure and TLR stimulation in human primary, monocyte-derived, and resident macrophages. A–C, primary THP-1 macrophages and microglial cells were inoculated with ZIKV (MR766, 1 m.o.i.) for 24 h, and CH25H mRNA expression was measured by qPCR. D and E, THP-1 macrophages and microglial cells were stimulated with a control (medium only) and a panel of TLR agonists (TLR1–TLR9) for 3 h, and CH25H mRNA expression was measured by qPCR. HPRT was used as an endogenous control to normalize the target mRNA, and the results are shown as means ± S.E. from triplicate samples. *, p < 0.0332; **, p < 0.0021; ****, p < 0.0001 (two-tailed unpaired Student's t test). Data represent results from three independent experiments with triplicate samples.

Figure 2.

Viral replication and the endosomal compartment are important for ZIKV-mediated CH25H. A, THP-1 Macrophages (Mφ) and microglial cells were exposed to either uninfected medium (Mock) or heat-inactivated or live ZIKV (MR766, 1 m.o.i.) for 24 h. RNA was then isolated and subjected to qPCR to determine CH25H expression. B, THP-1 macrophages and microglial cells were pretreated with DMSO or bafilomycin A₁ (5 μm) for 1 h and then exposed to ZIKV (MR766, 1 m.o.i.) for 24 h. RNA was then isolated and CH25H mRNA expression was measured by qPCR. HPRT or β₂M was used as an endogenous control to normalize the target mRNA, and the results are shown as means ± S.E. from triplicate samples. *, p < 0.0332; **, p < 0.0021; ****, p < 0.0001 (two-tailed unpaired Student's t test).

Figure 3.

CH25H expression occurs through TRIF and MyD88 signaling pathways. A and B, WT THP-1 macrophages were pretreated with either vehicle (Veh, DMSO) or BX795 (10 μm) for 1 h. In parallel, MyD88 KO THP-1 macrophages were also pretreated with DMSO for 1 h. Thereafter, both cell types were treated with LPS (A, 100 ng/ml) for 3 h or with MR766 (B, 1 m.o.i.) for 24 h. RNA was then isolated, and CH25H mRNA expression was measured by qPCR. C, THP-1 macrophages were pretreated with DMSO or TLR3 inhibitor (2.5 μm) for 1 h and then exposed to ZIKV (MR766, 1 m.o.i.) for 24 h. 24 h post-infection, cells were collected for CH25H analysis by qPCR. HPRT was used as an endogenous control to normalize the target mRNA, and the results are shown as means ± S.E. from triplicate samples. **, p < 0.0021; ****, p < 0.0001 (two-tailed unpaired Student's t test).

Figure 4.

Cytokine production with ZIKV exposure. A–F, THP-1 macrophages and microglial cells were inoculated with ZIKV (MR766, 1 m.o.i.) for 24 h. 24 h post-infection, cells were collected for selected pro-inflammatory cytokine and IFNs analysis by qPCR. HPRT or β₂M was used as an endogenous control to normalize the target mRNA. The results are shown as means ± S.E. from data pooled from two independent experiments with triplicate experiments. *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; ****, p < 0.0001 (two-tailed unpaired Student's t test). G–L, secretion of inflammatory cytokines was measured by ELISA using supernatants from THP-1 macrophages (G–I) and microglial cells (J–L) infected with MR766 (1 m.o.i.) for 48 h.

Figure 5.

Type I IFNs are not required for TLR- and ZIKV-mediated CH25H induction. A and B, THP-1 macrophages were pretreated with vehicle (DMSO) or cycloheximide (100 μm) for 1 h and then treated with poly(I:C) (10 μm) or LPS (100 ng/ml) for 3 h. RNA was collected for CH25H expression for qPCR (A). LPS-treated cells were used for Western blot analysis of IFNβ protein with GAPDH as a loading control (B). MW, molecular weight. C–E, THP-1 macrophages were first pretreated with isotype antibody (IgG), IFN receptor subunit 2 (IFNAR2)–neutralizing antibody (C and D), or type I IFN–neutralizing antibody mixture (E) for 1 h, followed by treatment with poly (I:C) (C and D) or LPS for 3 h or inoculated with ZIKV (E, MR766, 1 m.o.i.) for 24 h. CH25H or MX1 mRNA expression was measured by qPCR. F, microglial cells were pretreated with isotype antibody (IgG) or type I IFN–neutralizing antibody mixture for 1 h, followed by infection with ZIKV (MR766, 1 m.o.i.) for 24 h. CH25H mRNA expression was measured by qPCR. HPRT or β₂M was used as an endogenous control to normalize the target mRNA, and the results are shown as means ± S.E. from triplicate samples. *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002 (two-tailed unpaired Student's t test). NS, not significant; Mφ, macrophages.

Figure 6.

Induction of CH25H in human macrophages with pro-inflammatory cytokine stimulation. A, WT and MyD88 KO THP-1 macrophages were treated with either PBS or LPS for 3 h. 3 h post-treatment, culture supernatants from WT THP-1 macrophages were transferred to fresh MyD88 KO THP-1 macrophages for 3 h. Both the actual treatment and supernatant treatment were analyzed for CH25H mRNA expression by qPCR. B, THP-1 macrophages were treated with medium (Mock) or selected pro-inflammatory cytokines for 3 h, CH25H mRNA expression was measured by qPCR. HPRT or β₂M was used as an endogenous control to normalize the target mRNA, and the results are shown as means ± S.E. from triplicate samples. *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; ****, p < 0.0001 (two-tailed unpaired Student's t test).

Figure 7.

Potential CH25H transcription factor(s) induced by viral infection and cytokine stimulation. A and B, THP-1 macrophages (A) or PBMC monocyte derived macrophages (B) were inoculated with ZIKV (MR766, 1 m.o.i.) for 24 h. MW, molecular weight. C, THP-1 macrophages were treated with pro-inflammatory cytokines for 3 h. Cell lysates were analyzed for potential transcription factor induction and GAPDH protein by Western blotting.

Figure 8.

STAT1 regulates CH25H expression in human macrophages. A–C, THP-1 macrophages were pretreated with vehicle (DMSO) or JAK inhibitor I (1 μm) for 1 h and then treated with poly(I:C) (A, 10 μm) or LPS (100 ng/ml) for 3 h, with ZIKV (B, MR766, 1 m.o.i.) for 24 h, or with pro-inflammatory cytokines for 3 h (C). CH25H mRNA expression was measured by qPCR. D, THP-1 macrophages were treated with medium or LPS for 3 h, and ChIP analysis for STAT1 and ATF3 binding was performed at the CH25H promoter. *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; ****, p < 0.0001 (two-tailed unpaired Student's t test).