Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia

The following reagents were purchased from the indicated sources: Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), poly-L-lysine, Tris, bovine serum albumin (BSA), diphenylene iodonium (DPI), apocynin (APO, Sigma-Aldrich, St. Louis, MO); Iba1 (ionized calcium binding adaptor molecule 1) and Mac-1 antibodies (BD Biosceneces, San Diego, CA); acrylamide/bis-acrylamide gel (Bio-Rad, Hercules, CA); CDP-Star substrate (Applied Biosystems, Foster City, CA); K-Blue substrate (Neogen, Lexington, KY); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); anti-p38 and -extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK antibodies (Cell Signaling, Beverly, MA); recombinant murine interleukin (IL)-1β, tumor necrosis factor (TNF)-α, CCL2 CXCL10, anti-murine TNF-α, IL-1β, CCL2 and CXCL10 antibodies (R&D Systems, Minneapolis, MN); RNase inhibitor, SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA); DNase (Ambion, Austin, TX); random hexmer, and oligo (dT)_12-18 (Gene Link, Hawthorne, NY); SYBR^® Advantage^® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); 2', 7' -dichlorodihydrofluorescein diacetate (H₂DCFDA), SB203580 (an inhibitor of p38 MAPK), SB202474 (a negative control for SB203580), U0126 (an inhibitor of MAP kinase kinase [MEK]1/2, upstream of ERK1/2), and U0124 (a negative control for U0126) (EMD Chemicals, Gibbstown, NJ).

Female and male BALB/c mice, 8 to 10 weeks old, were purchased from Charles River (Wilmington, MA). These mice were housed in a specific pathogen free room (12-hr light-dark cycle) and had open access to a commercial diet and water. This study was approved by the University of Minnesota Institutional Animal Care, Use, and Research Committee.

Microglial cells were prepared as previously described [6, 15]. In brief, murine cerebral cortical brain tissues from 1 d-old mice were dissociated after a 30-min trypsinization (0.25%) and plated in 75-cm² Falcon culture flasks in DMEM containing 10% heat-inactivated FBS and antibiotics. The medium was replenished 1 and 4 days after plating. On day 12 of culture, floating microglial cells were harvested, plated into 96-well (4 × 10⁴ cells/well) or 12-well (1 × 10⁶ cells/well) plates, and incubated at 37°C. Purified microglial cell cultures were comprised of a cell population in which > 98% stained positively with Mac-1 and Iba-1 antibodies and < 2% stained positively with antibodies specific to glial fibrillary acidic protein (GFAP), an astrocyte marker.

HSV-1 strain 17 syn⁺ was propagated and titrated using plaque assay on rabbit skin fibroblasts (CCL68; American Type Culture Collection, Manassas, VA).

Production of intracellular ROS was measured using H₂DCFDA oxidation. Murine microglial cultures seeded (4 × 10⁴ /well) in 96-well plates or 4-well chamber slides were infected with HSV-1 (MOI = 2.5). At designated time points, cells were washed and incubated with HBSS (with Ca²⁺) containing H₂DCFDA (20 μM) for 45 min (avoiding light exposure). After incubation, cell culture plates were read using a fluorescence plate reader at Ex₄₈₅ and Em₅₃₀ or viewed and photographed under a fluorescence microscope. Each sample was run in triplicate and sample means were normalized to their respective controls (% of control).

One μg of total RNA extracted from microglia after treatment was treated with DNase and reverse transcribed to cDNA with oligo (dT)_12-18, random hexmer, dNTPs, RNase inhibitor and SuperScript™ III reverse transcriptase. Mixtures of diluted cDNA, primers and SYBR^® Advantage^® qPCR premix were subjected to real-time PCR (Stratagene, La Jolla, CA) according to manufacturer's protocol. Primer sequences were sense 5'-TGCTCGAGATGTCATGAAGG-3' and antisense 5'-AATCCAGCAGGTCAGCAAAG-3' for HPRT; sense 5'-GCCTCTTCTCATTCCTGCTTGT-3', antisense 5'- CACTTGGTGGTTTGCTACGAC-3' for TNF-α; sense 5'-AGACTTCCATCCAGTTGCCTTC-3' and antisense 5'-CATTTCCACGATTTCCCAGAG-3' for IL-6; sense 5'- AGGCTGGAGAGCTACAAGAGGA-3' and antisense 5'-GACCTTAGGGCAGATGCAGTTT-3' for CCL2; sense 5'-GTCATTTTCTGCCTCATCCTGCT-3' and antisense 5'-GGATTCAGACATCTCTGCTCATCA-3' for CXCL10. The relative mRNA expression levels were quantified using the 2^(-ΔΔCT) method [16] and were normalized to the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT; NM_013556).

In brief, 96-well ELISA plates pre-coated with goat or rabbit anti-mouse cytokine/chemokine antibody (2 μg/ml) overnight at 4°C were blocked with 1% BSA in PBS for 1 h at 37°C. After washing with PBS containing Tween 20 (0.05%), culture supernatants and a series of dilution of cytokines/chemokines (as standards) were added to wells for 2 h at 37°C. Anti-mouse cytokine/chemokine detection antibodies were added for 90 min followed by addition of anti-IgG horseradish peroxidase conjugate (1:10, 000) for 45 min. The chromogen substrate K-Blue was added at room temperature for color development which was terminated with 1 M H₂SO₄. The plate was read at 450 nm and cytokine/chemokine concentrations were extrapolated from the standard concentration curve.

Cell lysates collected after treatment were electrophoresed in 12% acrylamide/bis-acrylamide, electrotransferred onto nitrocellulose membrane and probed with antibodies for phospho-p38 (Thr180/Tyr182) and phospho-p44/42 (Thr202/Tyr204) MAP kinase followed by alkaline phosphatase-conjugated secondary antibodies with chemiluminescence detection using Kodak Image Station (Carestream Health (formerly Kodak), New Heaven, CT). Levels of phosphor-p38 (T180/Y182) and total p38 MAPK were measured using a Fast Activated Cell-based ELISA (FACE™), in-cell Western analysis according to the manufacturer's instructions (Active Motif, Carlsbad, CA).

Microglial cell cultures were pretreated with SB203580, SB202474, U0126 or U0124 for 1 h prior to viral infection followed by collection of cell culture supernatants for ELISA.

Data are expressed as mean ± SD or SEM as indicated. For comparison of means of multiple groups analysis of variance (ANOVA) was used followed by Scheffe's test.

To determine the role of redox responses in virus-induced cytokine and chemokine production, we first examined ROS production by HSV-stimulated microglia. Purified murine microglial cell cultures were infected with HSV at an MOI = 2.5. Virus-induced changes in intracellular ROS levels were assessed through loading the cells with the ROS fluorescence indicator H₂DCFDA and examination by fluorescence microscopy. In these studies, viral infection was found to induce rapid generation of microglial cell-produced ROS, as early as 3 h, with robust levels evident in most cells by 24 h p.i. (Figure 1). The concentration of H₂DCFDA used in these experiments (i.e., 20 μM) did not induce microglial cell toxicity as determined by MTT assay and trypan blue staining. In addition, MTT assay was used to check cell viability following viral infection and showed approximately 15% and 40% decreases at 24 and 48 h p.i., respectively.

We then went on to examine virus-induced ROS production over a time-course of infection. In these experiments, microglial cells were stimulated with HSV for the designated time, followed by quantification of H₂DCFDA oxidation using a fluorescence plate reader. Using this microplate assay, ROS levels in microglial cell cultures were found to be elevated by 24 h p.i., and reached maximal levels by 48 h (Figure 2A). We went on to investigate the effect of inhibition of NADPH oxidase on the production of this HSV-induced ROS. In these experiments, microglia were pretreated with the NADPH oxidase inhibitors DPI or APO for 1 h prior to viral stimulation. HSV-induced ROS production was significantly decreased by DPI in a concentration-dependent manner and by APO at 300 μM following the inhibition of NADPH oxidase (Figure 2B). The concentrations of DPI or APO used did not themselves induce microglial cell toxicity as determined by MTT assay and trypan blue staining.

We have previously reported that HSV stimulation of both human and murine microglial cells initiates robust cytokine and chemokine production [14, 15]. Data presented here demonstrate that ROS production by microglial cells occurs within 3 h following HSV infection. We've previously reported that cytokine and chemokine mRNA is first detectable using RT-PCR by 5 h p.i. and protein is first detectable by ELISA within 8 h p.i. [15]. The involvement of ROS in driving virus-induced expression of these immune mediators was investigated by pretreatment of microglial cells with DPI (0.03 - 1 μM) and APO (10 - 300 μM) and then using real-time RT-PCR to assess gene expression for select cytokines and chemokines. Treatment with either inhibitor of NADPH oxidase (i.e., DPI or APO) was found to inhibit TNF-α, interleukin (IL)-1β, CCL2, and CXCL10 mRNA expression at 5 h p.i. (Figure 3A-D). We went on to assess the involvement of NADPH oxidase and ROS in cytokine and chemokine production using ELISA to measure protein levels in cell culture supernatants. Corresponding to our findings at the mRNA level, both inhibitors of NADPH oxidase blunted cytokine (TNF-α and IL-1β) and chemokine (CCL2 and CXCL10) protein production in virus-infected microglial cultures (Figure 4A-D).

Activation of MAPKs plays an essential role in the cytokine response of microglial cells to inflammatory stimuli. p38 MAPK has recently been shown to be critical for the neurotoxic phenotype of monocytic cells following exposure to HIV gp120 [17]. For this reason, we examined whether HSV infection activated p38 and p44/42 MAPKs in our primary murine microglia. Using Western Blot, viral infection of primary microglial cells was found to stimulate phosphorylation of both kinases by 2 h p.i. (Figure 5A). These results were confirmed using a more quantifiable FACE in-cell Western assay over a 24 h time-course of infection. Using this assay, significant phosphorylation of p38 MAPK in response to viral infection was detected as early as 1 h p.i., with prolonged activation evident at 24 h p.i. (Figure 5B).

We went on to examine the effect of NADPH oxidase and ROS production on MAPK activation in response to viral infection. In these studies, treatment of microglial cells with either DPI or APO prior to viral infection blunted HSV-induced MAPK phosphorylation as detected using Western Blot at 2 h p.i. (Figure 6A). Additionally, FACE assay analysis at 2 h p.i. confirmed that either DPI or APO treatment significantly reduced phosphorylation of p38 MAPK (Figure 6B).

In the last set of experiments, we examined the involvement of these two ROS-driven MAPK signaling pathways in cytokine and chemokine production by microglia in response to viral infection. In these studies, inhibition of the p38 MAPK signaling pathway using SB203580 (0.1 to 10 μM) was found to suppress both cytokine (TNF-α and IL-1β) and chemokine (CCL2 and CXCL10) production (Figure 7). In contrast, inhibition of p44/42 MAPK signaling using U0126 (0.1 to 10 μM) inhibited cytokine (Figure 7A, B), but not chemokine production (Figure 7C, D). Additional assays tested whether MAPK inhibition affected HSV-induced ROS production itself. Data generated from these studies showed that the ERK1/2 (p44/p42) inhibitor U0126 partially suppressed ROS production by 11.1%, 18.1%, and 20.9%, at 0.1, 1.0, and 10 μM, respectively. Correspondingly, the p38 MAPK inhibitor SB203580 also partially suppressed ROS production by 16.3%, 21.1%, and 42.4%, at 0.1, 1.0, and 10 μM, respectively.