Background
Japanese encephalitis (JE) is an acute central nervous system (CNS) inflammatory disease caused by infection with Japanese encephalitis virus (JEV); a small, enveloped, plus-strand RNA virus belonging to the
Flaviviridae family. It is the leading cause of viral encephalitis in south-east Asia, India, and China where three billion people are at risk of contracting the disease, yet its pathogenesis remains poorly understood. While neurons are believed to be the primary target of JEV in the brain, a recent report has suggested that microglial cells can be directly infected with JEV [
1,
2]. Microglial cells are the resident immune cells of the CNS and have a critical role in host defense against invading pathogens. However, substantial evidence suggests that infection of microglia by JEV may actually lead to neuronal cell death through uncontrolled production of pro-inflammatory cytokines. Therefore, downregulation of cytokine production can serve to dampen the inflammatory response and can contribute to better virus clearance and increased protection against JEV [
3]. Indeed, inhibition of chronic neuroinflammation, particularly due to microglial activation, has been suggested to be a practical strategy in the treatment of neurodegenerative diseases [
4,
5].
Recently, a new class of regulatory RNAs, called microRNAs (miRNAs) have emerged that modulate immune response and play key regulatory roles in virus-host interactions. These miRNAs serve as universal regulators of differentiation, activation, and polarization of mammalian cells including microglia and macrophages in normal and diseased CNS [
6]. Thus, modulation of cellular miRNA expression during viral infection may be an important determinant of disease outcome [
7]. Recent reports suggested that a subset of miRNAs, termed as NeurimmiRs, co-exist in the brain and peripheral organs [
8,
9]. These miRNAs can affect both neuronal and immune functions and thus constitute important therapeutic targets for those diseases that affect both the immune system and brain functions. Among them, miR-155 and miR-146a are multifunctional and widely reported to modulate different stages of innate immune response during inflammation and infection [
10‐
12]. Thus, miR-155 and miR-146a were shown to increase in the microglial cells in response to stimulation with Lipopolysaccharides (LPS) and Polyinosinic-polycytidylic acid (poly(I:C), respectively, and they seem to play a fundamental role in the microglial inflammatory profile [
13,
14]. These miRNAs are also associated with interferon (IFN) signaling pathways [
15,
16]. Moreover, miR-155 and miR-146a not only modulate Toll-like receptors (TLRs)-mediated innate immune response, but also target complement regulatory proteins and facilitate complement activation [
17‐
19]. This phenomenon is very important to eliminate the virus from infected cells. Furthermore, both miR-146a and miR-155 have been shown to play an important role in viral infection. For example, Wu
et al.[
20] reported an increased dengue virus 2 (DENV2) replication in miR-146a overexpressing cells, whereas overexpression of miR-155 significantly suppressed human immunodeficiency virus (HIV) infection in activated macrophages [
21].
Since JEV is a neurotropic virus it is likely that NeurimmiRs play an important role in virus replication and immunopathology. Using a global miRNA array we have identified differentially expressed NeurimmiRs in human microglial cells during the course of JEV infection. Of these, we have focused our study on miR-155 and miR-146a and have investigated their effect on JEV replication and their role in the modulation of microglia-mediated innate immune response during JEV infection. For this purpose, in vitro studies were performed in JEV-infected human microglial CHME3 cells. Our results indicate that miR-155 induction might have a beneficial role for the host by limiting JEV replication through modulation of microglia-mediated innate immune responses.
Materials and methods
Cells, antibodies, miRNA mimics, and inhibitors
Human microglial cells (CHME3) were provided by the National Brain Research Centre, Manesar, India. Porcine stable kidney (PS) cell line was procured from National Centre for Cell Science, Pune, India. CHME3 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA) and PS cells in Eagle’s Minimal Essential Medium (MEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 μg/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Primary antibodies against MyD88, Ikkϵ, IRF8, p-STAT1, STAT1 and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology, Beverly, MA, USA. CD-45 antibodies were from BD Biosciences, San jose, CA, USA. Rabbit antibody against JEV NS1 protein was produced in-house. MicroRNA mimics and inhibitors were from Invitrogen, Carlsbad, CA, USA (Assay ID: MC12601, mimic for hsa-miR-155-5p; MH12601, inhibitor for hsa-miR-155-5p; MC10722, mimic for hsa-miR-146a-5p; MH10722, inhibitor for hsa-miR-146a-5p, mirVana® miRNA Mimic Control #1). NF-κB inhibitor ammonium pyrrolidine dithiocarbamate, and PI3K inhibitor LY294002 were from Sigma-Aldrich, Saint Louis, MO, USA.
Transfection of cells and virus infection
The P20778 strain of JEV was propagated in PS cells and titrated by plaque assay [
22]. Microglial cells were seeded in 6-well tissue culture plates at a density of 0.5 × 10
6 cells/well and transfected 24 hours later using Lipofectamine 2000® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For overexpression or inhibition studies, 25 pmol of a miRNA mimic or inhibitor, respectively, was transfected. Cells were washed with 1 × PBS after 24 hours transfection and infected with JEV at multiplicity of infection 5 (MOI = 5). Culture supernatant was collected for virus titration as plaque forming unit (PFU)/ml and cells lysate was used for protein and RNA studies at different times post-infection (pi).
RT2Profiler PCR array
The Human NFκB Signaling Targets PCR array (#PAHS-0225Z) (SA Biosciences/Qiagen, Hilden, Germany) was used to determine the profile of genes associated with the human innate and adaptive immune responses. Total RNA was extracted from JEV-infected and uninfected or specific mimic- and inhibitor-transfected CHME3 cells using RNeasy mini kit (Qiagen, Hilden, Germany) with inclusion of a DNase I treatment step. cDNA was prepared from 1 μg total RNA using a RT2 PCR array first strand kit (Qiagen, Hilden, Germany). Quantitative real-time PCR (qPCR) was performed with an ABI PRISM 7500 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.
Western blot analysis
Control and treated microglial cells were washed with 1 × PBS and lysed in cell lysis buffer (Sigma-Aldrich, Saint Louis, MO, USA) in the presence of protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). The concentration of the protein lysate was determined using the Bradford method. The protein sample (50 μg) was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was then blocked using 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20) for one hour at room temperature on a shaker. After blocking, the membrane was incubated with rabbit anti-human primary antibody overnight at 4°C with gentle shaking. After three washes of 10 minutes each with 1 × PBST, blots were incubated with anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibody for one hour with gentle shaking at room temperature. After three washes of the membrane for 10 minutes each with PBST the western blots were developed using chemiluminescence reagents (Santa Cruz Biotechnology, Dallas, Texas, USA). Afterwards membranes were stripped using stripping buffer (10% SDS, Tris-Cl pH-6.8, 0.8% β-mercaptoethanol) and re-probed with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies. The protein levels were normalized with the GAPDH levels.
Flow cytometry assay
Expression of CD-45 isoforms on CHME3 cells was examined by fluorescence activated cell sorter (FACS) analysis. Cells were fixed using 2% para-formaldehyde and stained with CD-45 antibody conjugated with Alexa-488 dye (BD Biosciences, San jose, CA, USA) as per the manufacturer’s instruction. Samples were analyzed on FACS Calibur cell analyser (BD Biosciences, San jose, CA, USA). Data were analyzed using FlowJo flow cytometry analysis software (Tree Star Inc, Ashland, OR, USA).
Quantitation of mRNA expression
Cells were lysed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA isolation and the total RNA was isolated using RNeasy kit (Qiagen, Hilden, Germany). RNA concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). RNA (1 μg) was reverse transcribed using prime script first-strand cDNA Synthesis kit (Takara Bio Inc. Otsu, Shiga, Japan) in a 20 μl reaction according to the manufacturer’s protocol. The expression of cellular genes was studied by qPCR with the fluorescent DNA-binding dye SYBR green (Power SYBR Green PCR master kit; Applied Biosystems, Foster City, CA, USA) by the real-time fluorescence detection method. JEV RNA was quantified as described earlier [
23]. Each quantitative PCR reaction was performed in triplicate and the mean threshold cycle (Ct) value for each sample was used for data analysis. The RNA transcript levels were normalized to that of GAPDH.
Quantitation of miRNA expression
For detection of mature miRNAs, 10 ng total RNA was reverse transcribed in vitro to cDNA using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. All miRNAs were assayed individually by TaqMan probe-directed real-time PCR (Reporter-FAM, Quencher-NFQ-MGB, Applied Biosystems, Foster City, CA, USA) using ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The following thermal cycling profile was used for the PCR analysis: 95°C for 15 minutes, 40 cycles at 94°C for 15 seconds, and 55°C for 30 seconds. Each qPCR reaction was performed in triplicate and the mean Ct value for each sample was used for data analysis. Expression levels of miRNAs were normalized to that of U6 snRNA.
Statistical analysis
All experiments were performed in triplicate. Gene expression profiling data were analyzed statistically using one-way analysis of variance (ANOVA) following Bonferroni’s multiple comparison tests. Data were presented as the mean ± SD; statistical significance of difference (P value) for two means was assessed using an unpaired Student’s t-test using the GraphPad Prism 5 software (GraphPad, San Diego, CA, USA), and P < 0.05 was considered significant.
Discussion
Microglia plays key roles in both innate and adaptive immune response in the CNS. In the mammalian immune system miRNAs control differentiation as well as innate and adaptive immune responses. A subset of these miRNAs (designated NeurimmiRs) notably affects both immune and neuronal functions. In this study, we have focused on two NeurimmiRs, miR-155 and miR-146a, and have studied their role in JEV replication and microglial activation during the infection. These two miRNAs are widely reported to have an immense effect on innate immune response in the context of various external stimuli (such as TLR ligands, virus, bacteria, and other microorganisms). However, very little is known about their role in innate immune modulation during neurotropic flavivirus infection. Here, we have used cultured human microglial cells to understand the effect of JEV infection on these two miRNAs. We have also studied how overexpression of these two miRNAs in microglial cells could affect JEV replication and how these miRNAs could modulate innate immune responses as well as microglial activation. We observed that the expression of miR-155 was enhanced in microglial cells during JEV infection. This may be due to the fact that miR-155 production is transiently under the control of
NF-κB which is activated during JEV infection and inflammation [
28,
32]. Since both these miRNAs are known to have a role in antiviral immunity, we hypothesized that upregulation of these miRNAs might play a role in controlling JEV infection. Interestingly, we found that JEV replication was significantly restricted in miR-155 overexpressing cells.
To gain an insight into the mechanism of how miR-155 overexpression suppressed JEV replication, we analyzed JEV genomic RNA
in silico with miRNA target prediction algorithms (PITA and RNA22) but found no potential target sites for miR-155. Hence, miR-155 is unlikely to target JEV RNA directly. We next examined
IFN-β in JEV-infected miRNAs overexpressing cells as inducible miR-155 in response to the vesicular stomatitis virus (an RNA virus) infection in macrophage was shown to induce type I IFN signaling and inhibit viral replication [
16]. However, to our surprise we found significant reduction in JEV-induced
IFN-β as well as downstream ISG mRNA expression in cells where miR-155 was overexpressed. This suggests that miR-155 uses different mechanisms to exert an antiviral effect against JEV in microglial cells. In fact, Swaminathan
et al.[
21] have shown that miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry, pre-integration events, leading to severely diminished HIV-1 infection. Thus, miR-155 can use different mechanisms for its antiviral effect depending upon the virus and the environmental stimuli generated during the particular virus infection of a certain cell type.
In order to understand reduced
IFN-β expression in JEV-infected miR-155 overexpressing cells, we checked the expression of IRFs. Among the several IRFs reported, we focused our study on
IRF8 as it plays a major role in
IFN signaling, response to infection, and maturation of myeloid lineages cells [
33].
IRF8 is also involved in the rapid induction of
IFN-β in human monocytes [
25]. Moreover,
IRF8 may activate a program of gene expression that transforms microglia into a reactive phenotype [
34]. In this study, we observed that JEV infection in microglia can induce
IRF8 expression, but the same expression was attenuated in miR-155 overexpressing cells and may be a possible reason for reduced
IFN-β production in miR-155 overexpressing cells. Interestingly, bioinformatics analysis predicted IRF8 as one of the potential targets for miR-155. We cloned 3’-UTR of
IRF8 in the luciferase reporter system for miRNA target validation but found no change in the luciferase readout in miR-155 overexpressing cells (data not shown). We have shown that
IRF8 is induced during JEV infection. Since JEV replication and titres are reduced in miR-155 overexpressing cells, it could result in reduced
IRF8 induction.
IRF8 is also involved in TLR-mediated
NF-κB activation [
26]. Since JEV-induced
IRF8 expression in miR-155 overexpressing cells was attenuated, we sought to understand its effect on
NF-κB pathway. For this we examined 84 genes by RT-PCR array whose expression and function, either directly or indirectly, depend on
NF-κB activation. We found several genes, including
IFN-β,
MyD88, STAT1, PTGS2, and
IL-12B, which are induced by JEV and show attenuated expression in miR-155 overexpressing cells. Recently,
CCR5 receptor expression in a mouse model of JE was reported to play an important role in recovery as well as promote host survival against JEV [
27]. Interestingly, increased
CCR5 expression was also observed in our cell culture based study. Thus, modulation of NF-κB mediated signaling pathway genes by JEV-induced miR-155 expression might play a role in reduced JEV replication in microglial cells.
Specific induction of active SHP2 phosphatase dephosphorylates IRF8, which in turn becomes an active repressor and down-regulates TLR-mediated gene expression [
35]. In hematopoietic cell lineage
CD45 (a Src-homology 2 domain (SH2)-containing protein tyrosine phosphatase) plays an important role in regulating cytokine receptor mediated signal [
36]. Analysis of microglia
ex vivo revealed that IRF8-deficient microglia had significantly increased levels of
CD45[
37]. Thus attenuated
IRF8 expression in miR-155 overexpressing cells may result in enhanced
CD45 expression on microglial cells.
Following an infection, resting microglial cells can get activated into M1 or M2 phenotypes. The M1 phenotype is a pro-inflammatory state and induces neuropathology, whereas M2 is anti-inflammatory state that could have a neuroprotective role [
36]. It is reported that
CD45 can negatively regulate CD40L-CD40-induced microglial M1 activation; an effect leading to the promotion of the M2 phenotype. Moreover, this
CD45-mediated activation state appears to decrease harmful cytokine production [
36]. In this study, we observed an increased
CD45 expression in JEV-infected miR-155 overexpressing cells and relatively reduced
p-STAT1 expression. Further, mRNA expression for pro-inflammatory cytokines
IL-1β and
TNF-α was reduced in miR-155 overexpressing cells.
CD45 has also been shown to down-regulate NF-κB, an important mediator of pro-inflammatory cytokines [
38]. Thus, increased
CD45 expression in JEV-induced miR-155 expressing cells may explain reduced expression of NF-κB pathway genes in our study. Taken together, these data show that miR-155 induction can modulate the JEV-induced microglial activation to a state that may be beneficial to the host. Further
in vivo study is needed to clarify this issue.
Complement activation is considered to be an important component of innate immune response against invading pathogens.
CFH is one of the regulators which negatively regulate the complement activation. Viruses can utilize
CFH to evade the innate immune response. The non-structural protein NS1 of the West Nile virus, a flavivirus, inhibits complement activation by binding to
CFH[
39].
CFH can be induced by
NF-κB activation and can be regulated through miRNAs [
17]. Interestingly, both miR-155 and miR-146a have the same target sequence in the 3’-UTR of
CFH and this has been experimentally proven for both the miRNAs [
17]. We have shown that JEV infection can induce
CFH expression in human and mouse microglial cells and this induction is attenuated in miR-155 overexpressing cells. Therefore, it is possible that miR-155 expression in JEV-infected microglial cells inhibits
CFH expression, which in turn may benefit the host by facilitating complement activation against JEV.
In a study published earlier this month, Thounaojam
et al. [
40] showed an up-regulation of miR-155 in mouse microglial cells (BV-2) and in the mouse and human brain during JEV infection. They suggested that miR-155 had a pro-inflammatory role as its inhibition decreased
TBK-1, IRF3/7, and
NF-κB phosphorylation both in BV-2 cells as well as in the mouse brain. These results point to a role for miR-155 that is opposite to what we have observed in our study. A major difference between these two studies is that while results reported by Thounaojam
et al.[
40] are derived from mouse BV-2 cells and the mouse brain where various different kinds of cells may get infected with JEV, our results are derived entirely from
in vitro cultured human microglial cells CHME3. Additionally, the GP78 strain of JEV used by Thounaojam
et al.[
40] is a slow growing virus, both in cultured cells and the mouse brain, with lesions in the virus-cell fusion process [
22]. Another reason for these differences may be related to the smaller increase (approximately 4-fold) of miR-155 during JEV infection in BV2 cells [
40], compared with the super maximal concentration in CHME3 cells (approximately 100-fold) that occurred during miR-155 overexpression in the present study. Several reports have suggested that, depending on the expression level, miR-155 can modulate cellular functions by targeting genes in different pathways. Ceppi
et al.[
41] showed that low level miR-155 expression enables the activation of
p38 MAPK pathway, favoring
IL-1β expression, which induces inflammation in an autocrine manner. However, the same pathway was inhibited when the miR-155 expression level went up significantly, ultimately reflecting an altered immune profile. Similar observations were made by Xiao
et al.[
42] who reported a 3-fold induction of miR-155 and pro-inflammatory cytokine responses during
Helicobacter pylori infection, whereas the overexpression of miR-155 negatively regulated the pro-inflammatory responses.
Intracellular signaling pathways that are concomitantly activated by the same stimulus often interact with one another through a cross regulatory feedback mechanism. The miR-155 is a multifunctional microRNA and it can modulate inflammatory responses in both a positive and negative way [
40‐
48]. Besides its positive role in
NF-κB activation and subsequent pro-inflammatory response, accumulating evidence has demonstrated that it can constitute a negative feedback loop in the
NF-κB signaling pathway by targeting multiple key proteins, which ultimately leads to repression of, or at least the limitation of
NF-κB activation in response to viral or microbial stimuli [
41,
42,
45‐
48]. Therefore, miR-155 could modulate inflammation depending on various factors including its expression level, the cell type, and the environmental stimuli.
Overall our study suggests that miR-155 modulation can act on multiple levels to control JEV infection of microglial cells and induce innate immune responses that may be beneficial to the host. It can enhance CD45 expression, reduce pro-inflammatory cytokines and CFH expression by targeting several key genes, and suppress JEV replication in microglial cells. These data point to miR-155 playing an important role in modulating JEV-induced microglial activation that may be beneficial in limiting JEV infection in the host. Additional studies, both in vitro and in vivo, are needed to further understand the role of miR-155 during JEV infection in switching microglial activation towards the neuroprotective state.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AB was responsible for experimental design, data analysis, and drafting the manuscript. SP, BK, and PJ performed the RNA extraction, PCR array, RT-PCR, miRNA assay, western blot, and FACS. SR and BK performed the virus preparation, cell culture, and transfection and virus infection experiments. SV and AB conceived the idea, supervised the experiments, and participated in editing the manuscript. All authors have read and approved the final manuscript.