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, 16 (1), 248

HSV-1 Triggers Paracrine Fibroblast Growth Factor Response From Cortical Brain Cells via Immediate-Early Protein ICP0

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HSV-1 Triggers Paracrine Fibroblast Growth Factor Response From Cortical Brain Cells via Immediate-Early Protein ICP0

Niko Hensel et al. J Neuroinflammation.

Abstract

Background: Herpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells.

Method: We employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells.

Results: Astrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism.

Conclusions: HSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.

Keywords: Akt; Cortex; ERK; FGF; Fibroblast growth factors; HSV-1; ICP0; Neurotrophic factors; Signaling.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Characterization of HSV-1-infected primary cortical neurons (PCC). a Murine PCCs were infected with HSV-1(17+)LoxpCMVGFP (MOI 10) at DIV5 and compared with mock-infected control cells 16 hpi. Cells were stained against the neuronal marker βIII-tubulin (βIII-tub), the astrocytic marker glial fibrillary acidic protein (GFAP), the oligodendrocyte transcription factor (olig-2), and allograft inflammatory factor (Aif1/Iba-1) as a marker for microglia. b Cell type composition of the mock infected PCCs. c Percentage of HSV-1 positive cells 6 and 16 hpi defined for each cell type. Bars show mean ± SEM (n = 3) with a two-way ANOVA and a Holm-Sidak’s multiple comparison test (**p < 0.01, ***p < 0.001 compared to 6 hpi astrocytes, ###p < 0.001 compared to 16 hpi astrocytes). d The astrocytes in the PCCs were HSV-1(17+)LoxpCMVGFP infected (MOI 10) and analyzed 6 hpi and 16 hpi via GFAP staining. eg GFAP positive astrocytes were characterized using the automated cell image analysis software CellProfiler. e The area of HSV-1 negative and HSV-1-positive astrocytes was measured within mock control and HSV-1-infected PCCs. f Compactness of infected and non-infected astrocytes. g Classification of HSV-1 positive and HSV-1 negative astrocytes depending on the area of the cell body related to the total astrocyte area (large > 1000 μm2, medium 1000 μm2 ≥ × ≤ 500 μm2, small < 500 μm2). Sidak’s multiple comparison tests refer to mock-infected control astrocytes of the same size-class. hj mRNA levels of A1/A2 markers were quantified by qRT-PCR in PCCs 6 and 16 hpi. All bars show mean ± SEM (n = 3) with a two-way ANOVA (eg) and a one-way ANOVA (hj) followed by Sidak’s multiple comparison test (****p < 0.0001, **p < 0.01, *p < 0.05)
Fig. 2
Fig. 2
Upregulated fibroblast growth factors (FGFs) in response to HSV-1 infection. a FGF mRNAs were quantified by an initial qRT-PCR screening in control and HSV-1(17+)Lox-infected PCCs (MOI 10) 0, 2, 4, 6, or 8 hpi. 0 hpi cells were inoculated with HSV-1 for 30 min without any incubation in culture medium at 37 °C and 5% CO2. Instead, cells were immediately lyzed. be Validation of the screening results with an increased number of biological independent replicates. Bars show mean ± SEM with n = 2 for a, and n = 6 for be. Student’s t test with (**p < 0.01, *p < 0.05)
Fig. 3
Fig. 3
FGF-expression and paracrine induction of the ERK-pathway in response to HSV-1 infection of PCCs. a Mock-infected control PCCs and HSV-1(17+)LoxpCMVmCherry infected cells (8 hpi, MOI 10) were stained for GFAP and FGF-9. b HSV-1(17+)Lox infected PCCs were infected and the conditioned medium was collected 30 mpi, 4 hpi, and 8 hpi, while the supernatant media of control cells collected 8 h post mock-infection. Viral particles were removed by filtration. Non-infected PCCs were starved 2 h prior to conditioned medium incubation reducing pathway activities to a basal level. Subsequently, starved PCCs were incubated for 30 min with conditioned medium and immediately lyzed for Western blot analysis. c Representative Western blots of infected source cells and non-infected target cells treated with conditioned medium. d, e Densitometric analysis of ERK and Akt phosphorylation in HSV-1-infected source PCCs. f, g Densitometric analysis of ERK (e) and Akt (f) phosphorylation in non-infected PCCs treated with conditioned medium. Bars show mean ± SEM (n = 5) with a one-way ANOVA and a Holm-Sidak’s multiple comparison test (**p < 0.01, *p < 0.05). h Conditioned media of HSV-1(17+)Lox infected and control PCCs were collected 20 hpi, filtered and supplemented with either FGFR-inhibitor PD173074 (200 nM) or inhibitor vehicle DMSO. Target PCCs were pre-incubated for 2 h with starvation medium containing PD173074 or DMSO before they were incubated with the conditioned media for 30 min. i Representative phospho-ERK Western blots of cells treated with conditioned media. j Densitometric analysis of ERK phosphorylation in PCCs stimulated with conditioned and supplemented media. Bars show mean ± SEM (n = 5) with one-way ANOVA (+p < 0.05) and Fisher’s LSD post-test with *p < 0.05 and ns indicating non-significance for comparisons between control and HSV-1 CM treated cells and #p < 0.05 and ns# for comparisons of PD173074 treated cells with their DMSO control, respectively
Fig. 4
Fig. 4
FGF-induction is specific to CNS cells and depends on a functional HSV-1. a FGF-4 expression of PCCs, astrocytes, and murine mammary gland epithelial cell line (C127i) infected with HSV-1(17+)Lox (MOI 10). b FGF-4 expression of mock-infected control primary human keratinocytes compared to HSV-1(17+)Lox infected primary human keratinocytes (MOI 10, 6 hpi). Mean ± SEM (n = 3); two-way ANOVA with Holm-Sidak’s multiple comparison test (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). c FGF-4 expression in PCCs infected with HSV-1(17+)Lox (MOI 10), Theiler’s murine encephalomyelitis virus (TMEV, BeAn, MOI 10) or treated with dithiothreitol (DTT, 1 mM) 6 h after infection or treatment. d Fold change of FGF-4 mRNA in PCCs infected with untreated or UV-inactivated HSV-1(17+)Lox. All bars show mean ± SEM (n = 3) with Student’s t test (b), two-way ANOVA (a), and one-way ANOVA (cd) followed by a Holm-Sidak’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)
Fig. 5
Fig. 5
ICP0 deficient HSV-1 is not able to induce an FGF-response. a FGF-4 mRNA expression of PCCs which were infected with deletion mutants for neurovirulence factor ICP34.5 or RNA binding protein US11 (MOI 10, 6 hpi). b PCCs were infected with HSV-1 (KOS) as well as HSV-1 KOS knockout strains lacking ICP0, 4, 22, and 27 (MOI = 10, 6 hpi). ce FGF transcripts of HSV-1 (KOS) infected PCCs compared to the knockout KOS strain without ICP0 (MOI = 10, 6 hpi). All bars show mean ± SEM (n = 3) with a one-way ANOVA followed by a Holm-Sidak’s multiple comparison test (*p < 0.05, ***p < 0.001)

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