Cellular microRNA-155 Regulates Virus-Induced Inflammatory Response and Protects against Lethal West Nile Virus Infection

Abstract

West Nile virus (WNV) is a flavivirus that has disseminated globally as a significant cause of viral encephalitis in humans. MircoRNA-155 (miR-155) regulates various aspects of innate and adaptive immune responses. We previously reported that WNV infection induces upregulation of miR-155 in mice brains. In the current study, we demonstrate the critical role of miR-155 in restricting the pathogenesis of WNV infection in mice. Compared to wild-type (WT) mice, miR-155 knockout mice exhibited significantly higher morbidity and mortality after infection with either a lethal strain, WNV NY99, or a non-lethal strain, WNV Eg101. Increased mortality in miR-155^-/- mice was associated with significantly high WNV burden in the serum and brains. Protein levels of interferon (IFN)-α in the serum and brains were higher in miR-155^-/- mice. However, miR-155^-/- mice exhibited significantly lower protein levels of anti-viral interleukin (IL)-1β, IL-12, IL-6, IL-15, and GM-CSF despite the high viral load. Primary mouse cells lacking miR-155 were more susceptible to infection with WNV compared to cells derived from WT mice. Besides, overexpression of miR-155 in human neuronal cells modulated anti-viral cytokine response and resulted in significantly lower WNV replication. These data collectively indicate that miR-155 restricts WNV production in mouse and human cells and protects against lethal WNV infection in mice.

Keywords: West Nile virus; immune response; inflammatory cytokines and chemokines; miR-155; micro-RNAs; virus replication.

Figure 1

Clinical scores and survival analysis of West Nile virus (WNV) NY99 and WNV Eg101 infected WT and miR-155^−/− mice. (A,B) WT and miR-155^−/− mice were monitored twice daily for clinical signs as described in the materials and methods. Error bars represent SEM, * p < 0.05, ** p < 0.001. (C,D) The statistical differences in the survival of WT and miR-155^−/− mice were significant for both WNV NY99 and WNV Eg101 (n = 20 per group for WNV NY99 and n = 12 per group for WNV Eg101). ** p < 0.001.

Figure 2

Virus load in the serum and brains of WNV-infected miR-155^−/− and WT mice. (A,B) Virus titers (plaque-forming units (PFU)/mL) were assessed in the serum at days 2 and 4 after WNV NY99 or WNV Eg101 inoculation by plaque assay. (C) Virus titers (PFU/g of tissue) were measured in the brains at day 8 after inoculation with WNV NY99 or WNV Eg101. Each data point represents an individual mouse. The solid horizontal lines signify the median. * p < 0.05.

Figure 3

Levels of interferon (IFN)-α in WT and miR-155^−/− mice following WNV NY99 infection. (A) Protein levels of IFN-α were assessed in the mice serum at day 3 after inoculation and expressed as pg/mL of serum. (B) IFN-α levels were measured in brain homogenates at day 8 after inoculation and expressed as pg/g of brain tissue. Error bars represent SEM (n = 6–8 mice per group). * p < 0.05.

Figure 4

Serum protein levels of cytokines in the miR-155^−/− and WT mice following WNV NY99 infection. Protein levels of IL-1β, IL-6, IL-12, IL-10, IL-15, TNF-α, GM-CSF, and G-CSF were assessed in the serum by luminex assay. Data represent the mean concentration (pg/mL) ± SEM (n = 6–8 mice per group). * p < 0.05.

Figure 5

Serum chemokines levels in the WNV NY99-infected miR-155^−/− and WT mice. Protein levels of CCL4, CCL5, CXCL9, and CXCL10 were assessed in the serum by luminex assay. Data represent the mean concentration (pg/mL) ± SEM (n = 6–8 mice per group). * p < 0.05.

Figure 6

Virus titers in WNV-infected mouse embryonic fibroblasts (MEFs) and bone marrow-derived macrophages (BMDMs) isolated from miR-155^−/− and WT mice. (A–D) MEFs and BMDMs were infected as described in the methods and viral titers in the culture supernatants were assessed by plaque formation assay. The results expressed as PFU/mL ± SEM from three independent experiments conducted in duplicate. ** p < 0.001.

Figure 7

miR-155 mimic inhibits WNV replication in human neuroblastoma cells. (A) SK-N-SH cells were transfected with miR-155 mimic or control mimic. Cells were infected with WNV NY99 at a MOI of 1. Viral titers in the cell culture supernatants were assessed by plaque assay and expressed as PFU/ml ± SEM. (B) mRNA levels of IL-1β, IL-6, and IL-15 genes were determined using qRT-PCR at 24 h after infection, and the fold change in infected cells compared to corresponding controls was calculated after normalizing to the GAPDH gene. Data represents the mean ± SEM, representing two independent experiments. * p < 0.05, ** p < 0.001.