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. 2009 Dec;83(23):12204-14.
doi: 10.1128/JVI.01103-09. Epub 2009 Sep 23.

Regulation of proinflammatory cytokine expression in primary mouse astrocytes by coronavirus infection

Dongdong Yu  1 , Hongqing Zhu, Yin Liu, Jianzhong Cao, Xuming Zhang
Affiliations

Regulation of proinflammatory cytokine expression in primary mouse astrocytes by coronavirus infection

Dongdong Yu et al. J Virol. 2009 Dec.

Abstract

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), are differentially induced in primary mouse astrocytes by mouse hepatitis virus strain A59 (MHV-A59) and MHV-2. However, the signaling events that trigger TNF-alpha and IL-6 induction in these cells upon MHV infection remain unknown. In this study, we explored the potential signaling events. We found that induction of TNF-alpha and IL-6 occurred as early as 2 h postinfection and was completely dependent on viral replication. Using inhibitors specific for three mitogen-activated protein kinases, we showed that induction of TNF-alpha and IL-6 by MHV-A59 infection was mediated through activation of the Janus N-terminal kinase signaling pathway, but not through the extracellular signal-regulated kinase or p38 signaling pathway. This finding was further confirmed with knockdown experiments using small interfering RNAs specific for Janus N-terminal kinase. Interestingly, while nuclear factor kappaB (NF-kappaB), a key transcription factor required for the expression of proinflammatory cytokines in most cell types, was activated in astrocytes during MHV-A59 infection, disruption of the NF-kappaB pathway by peptide inhibitors did not significantly inhibit TNF-alpha and IL-6 expression. Furthermore, experiments using chimeric viruses demonstrated that the viral spike and nucleocapsid proteins, which play important roles in MHV pathogenicity in mice, are not responsible for the differential induction of the cytokines. These results illustrate the complexity of the regulatory mechanism by which MHV induces proinflammatory cytokines in primary astrocytes.

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Figures

FIG. 1.
FIG. 1.
Differential induction of TNF-α and IL-6 in primary mouse astrocytes by infection with MHV-A59 and MHV-2. (A) (Right) The purity of isolated primary mouse astrocytes was determined by immunofluorescence staining with an antibody specific for GFAP. (Left) Phase-contrast image showing the same field as in fluorescence staining. (B and C) Induction of TNF-α (B) and IL-6 (C) in primary astrocytes by MHV infection. Cells were infected with MHV-A59 or MHV-2 at an MOI of 5 or mock infected as a control. The culture supernatants were collected at 24 h p.i. The amounts of TNF-α (B) and IL-6 (C) proteins in the supernatants were determined using ELISA kits. The results are expressed as the mean number of picograms per milliliter for three independent experiments. The error bars indicate standard deviations of the means. The asterisks indicate the statistical significance between the pairs (P < 0.05). (D) Virus production in primary mouse astrocytes. Virus infection was carried out as for panels B and C, and virus titers were determined at 24 h p.i. by plaque assay on DBT cells. The results are expressed as the mean number of PFU per milliliter for three independent experiments. The error bars indicate standard deviations of the means. (E and F) Dependence of TNF-α and IL-6 induction on virus replication. Primary astrocytes were infected with live or UV-inactivated MHV-A59 at an MOI of 5 or mock infected. At 24 h p.i., the culture supernatants were collected to determine TNF-α (E) or IL-6 (F) protein amounts by ELISA as for panels B and C.
FIG. 2.
FIG. 2.
Kinetics of induction of TNF-α and IL-6 in primary astrocytes by MHV-A59. Primary astrocytes were infected with MHV-A59 at an MOI of 5. The cells and culture medium were separately collected at 0, 1, 2, 3, 4, 7, and 12 h p.i. for mRNA (A and B) and protein (C and D) detection. (A and B) Kinetics of TNF-α and IL-6 mRNA induction. Intracellular RNAs were isolated, and the amounts of TNF-α and IL-6 mRNA were determined by qRT-PCR. The results are expressed as the mean number of picograms of cDNA normalized to β-actin for three independent experiments. The error bars indicate standard deviations of the means. (C and D) Kinetics of TNF-α and IL-6 protein induction. The amounts of TNF-α and IL-6 protein in the supernatants were determined with an ELISA kit. The results are expressed as the mean number of picograms per milliliter for three independent experiments. The error bars indicate standard deviations of the means.
FIG. 3.
FIG. 3.
Characterization of signaling pathways involved in TNF-α and IL-6 induction by MHV-A59 infection. (A and B) Primary astrocytes were treated with the MEK inhibitor UO126 (50 μM), the control inhibitor UO124 (50 μM), the p38 inhibitor SB203580 (20 μM), the control inhibitor SB202474 (20 μM), or the JNK inhibitor SP600125 (40 μM) or mock treated with dimethyl sulfoxide (−) for 1 h prior to and throughout infection. The cells were infected with MHV-A59 at an MOI of 5. The culture supernatants were collected at 24 h p.i. to determine TNF-α (A) and IL-6 (B) amounts and the virus titer (E). The amounts of TNF-α and IL-6 proteins were determined with an ELISA kit. The results are expressed as the mean number of picograms per milliliter for three independent experiments. The error bars indicate standard deviations of the means. The asterisks indicate statistical significance between the pairs (P < 0.05). (C and D) Effects of specific MAP inhibitors on MEK and p38 signaling. Primary astrocytes were treated with UO126/UO124 or SB203580 (SB80)/SB202474 (SB74) at the indicated concentrations and were infected with MHV-A59 at an MOI of 5. The cell lysates were used for detection of total (T) or phosphorylated (P) MEK and p38 in Western blots using specific antibodies. Cells infected with virus alone (lanes Virus) were used as a positive control for potential activation of the MAP kinase signaling pathways, while uninfected and untreated cells were used as a negative control for basal-level expression (lane Mock). (E) Virus titers were determined by virus plaque assay on DBT cells and are expressed as the mean number of PFU per milliliter for three independent experiments. The error bars indicate standard deviations of the means.
FIG. 4.
FIG. 4.
Differential activation of the JNK signaling pathway by infections with MHV-A59 and MHV-2. (A) Primary astrocytes were infected with MHV-A59 or MHV-2 at an MOI of 5 or mock infected. Cells were then collected at 24 h p.i. Phosphorylated (P) and total (T) JNK and phosphorylated c-Jun were detected by Western blotting with phosphorylation-specific antibodies. β-Actin was used as an internal control. (B) Kinetics of JNK pathway activation by MHV-A59 infection. Primary astrocytes were infected with MHV-A59 at an MOI of 5. The cell lysates were collected at 0, 1, 2, 3, 4, 5, 6, 12, 18, and 24 h p.i. Phosphorylated and total JNK and phosphorylated c-Jun were detected by Western blotting as for panel A.
FIG. 5.
FIG. 5.
TNF-α induction by MHV-A59 infection is inhibited by JNK siRNAs. (A) Primary astrocytes were transfected with siRNAs to JNK1 and JNK2 singly or in combination or were transfected with a nonspecific siRNA (GloSiRNA). The cells were then infected with MHV-A59 at an MOI of 5 or mock infected. The cell lysates were collected at 24 h p.i. for detection of JNK, phosphorylated (P) c-Jun, and total (T) c-Jun by Western blotting using the respective antibodies. (B) Culture supernatants were collected at 24 h p.i. for detection of TNF-α protein by ELISA. The results are expressed as the mean number of picograms per milliliter for three independent experiments. The error bars indicate standard deviations of the means. The asterisks indicate a statistical significance between the pairs (P < 0.05).
FIG. 6.
FIG. 6.
Virus replication is required for JNK activation. Primary astrocytes were infected with live or UV-inactivated MHV-A59 at an MOI of 5 or mock infected. At 24 h p.i., the cell lysates were isolated and used for detecting phosphorylated JNK (P-JNK) and c-Jun (P-c-Jun) or total JNK (T-JNK) by Western blot analysis using specific antibodies as described in the legend to Fig. 5. β-Actin was used as an internal control.
FIG. 7.
FIG. 7.
Activation of TNF-α by MHV-A59 infection. (A) Schematic diagram showing the sequence elements of the murine TNF-α (mTNF-α) promoter and the binding sites for transcription factors NF-κB (κB), CRE, AP-1, and Sp1. (B) Nuclear translocation of NF-κB following MHV-A59 infection. Cells were infected with MHV-A59 at an MOI of 5 for 18 h (right) or mock infected (left). The cells were fixed with 4% paraformaldehyde and stained with primary anti-NF-κB p65 polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. The images were taken with a digital camera under a fluorescence microscope. The arrows indicate the nuclear translocation. (C) Assay for NF-κB activation. Primary astrocytes were infected with MHV-A59 at an MOI of 5 or mock infected. At 18 h p.i., nuclear extracts were isolated and NF-κB activity was determined with the TransAM NF-κB p65 kit as described in Materials and Methods. The results are expressed as the mean absorbance at 450 nm (OD450) for three independent experiments. The error bars indicate standard deviations of the mean. The asterisk indicates statistical significance between the pair (P < 0.05).
FIG. 8.
FIG. 8.
MHV-induced NF-κB activation is mediated through the JNK signaling pathway. Primary astrocytes were pretreated with the NF-κB inhibitor peptide Ser276 (Ser) at 50 μM or with a nonspecific inhibitor (NS) or were untreated (−) in the presence (SP) or absence (mock) of the JNK inhibitor SP600125. The cells were then infected with MHV-A59 at an MOI of 5. At 24 h p.i., cell nuclear extracts were prepared and NF-κB activity was determined with the TransAM NF-κB p65 assay kit, as described in Materials and Methods. The results are expressed as the mean absorbance at 450 nm (OD450) for three independent experiments. The error bars indicate standard deviations of the mean. The asterisks indicate statistical significance between the pairs (P < 0.05).
FIG. 9.
FIG. 9.
Role of NF-κB activation in the induction of TNF-α and IL-6 in primary astrocytes by MHV infection. (A) Primary astrocytes were pretreated with the NF-κB inhibitor SC3060 or NF-κB p65 (Ser276)-inhibitory peptide at a concentration of 50 μM or treated with a control peptide (NS). The cells were then infected with MHV-A59 at an MOI of 5 or mock infected (MI) as a control. The cell nuclear lysates were extracted at 24 h p.i. for determination of NF-κB activity with a TransAM NF-κB p65 kit. (B and C) NF-κB activity was expressed as the mean absorbance at 450 nm (OD450) in three independent experiments. The error bars indicate standard deviations of the means. The culture supernatants were collected at 24 h p.i. to measure TNF-α (B) and IL-6 (C) protein levels by ELISA. The amounts of TNF-α and IL-6 were expressed as mean numbers of picograms per milliliter in three independent experiments. The error bars indicate standard deviations of the means. The asterisks indicate statistical significance between the pairs (P < 0.05).
FIG. 10.
FIG. 10.
The S and N genes of MHV-A59 are not responsible for the induction of TNF-α. (A) Schematic diagram of the MHV genome highlighting the differences in S and N genes in the recombinants Penn-98-1 and icA59/2N with respect to the parental MHV-A59 and MHV-2. (B and C) Induction of TNF-α and IL-6 in primary astrocytes by MHV infection. Primary astrocytes were infected with the recombinant viruses Penn-98-1 and icA59/2N or the parental viruses MHV-A59 and MHV-2 at an MOI of 5, and the cell culture supernatants were collected at 24 h p.i. The protein levels of TNF-α and IL-6 were determined by ELISA and are expressed as mean numbers of picograms per milliliter in three independent experiments. The error bars indicate standard deviations of the means. The asterisks indicate statistical significance between the pairs (P < 0.05).
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