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IFNβ Protects Neurons From Damage in a Murine Model of HIV-1 Associated Brain Injury

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IFNβ Protects Neurons From Damage in a Murine Model of HIV-1 Associated Brain Injury

Victoria E Thaney et al. Sci Rep.

Abstract

Infection with human immunodeficiency virus-1 (HIV-1) causes brain injury. Type I interferons (IFNα/β) are critical mediators of any anti-viral immune response and IFNβ has been implicated in the temporary control of lentiviral infection in the brain. Here we show that transgenic mice expressing HIV-1 envelope glycoprotein 120 in their central nervous system (HIVgp120tg) mount a transient IFNβ response and provide evidence that IFNβ confers neuronal protection against HIVgp120 toxicity. In cerebrocortical cell cultures, neuroprotection by IFNβ against gp120 toxicity is dependent on IFNα receptor 1 (IFNAR1) and the β-chemokine CCL4, as IFNAR1 deficiency and neutralizing antibodies against CCL4, respectively, abolish the neuroprotective effects. We find in vivo that IFNβ mRNA is significantly increased in HIVgp120tg brains at 1.5, but not 3 or 6 months of age. However, a four-week intranasal IFNβ treatment of HIVgp120tg mice starting at 3.5 months of age increases expression of CCL4 and concomitantly protects neuronal dendrites and pre-synaptic terminals in cortex and hippocampus from gp120-induced damage. Moreover, in vivo and in vitro data suggests astrocytes are a major source of IFNβ-induced CCL4. Altogether, our results suggest exogenous IFNβ as a neuroprotective factor that has potential to ameliorate in vivo HIVgp120-induced brain injury.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Transient IFNβ expression is accompanied by up-regulation of interferon-stimulated genes in HIVgp120tg mouse brains.
Total brain RNA was extracted from age-matched HIVgp120tg and wild type control, male animals and qRT-PCR analysis was used to assess differential regulation of the indicated genes between the age-groups: (a) IFNβ; (b) IFNα; (c) MX1; (d) CXCL11; (e) CXCL10; (f) CCL2. GAPDH was used as internal control, and representative results are shown as fold-change (FC) differences compared to the WT control within the age group. Data are shown as mean ± s.e.m. (***P ≤ 0.001, **P ≤ 0.01 by ANOVA with Fisher’s PLSD post hoc test). n = 3–4 animals per group/genotype.
Figure 2
Figure 2. IFNβ mediates neuroprotection against HIV gp120-induced injury.
Rat cerebrocortical cultures were treated with gp120 from HIV-1 strain BaL (200 pM) for 24 h in the presence or absence of different IFNβ concentrations (500 or 5,000 U/ml) or BSA/PBS (0.001% final concentration) as vehicle control. Following the incubation the cells were fixed and permeabilized, and neurons were immunolabeled for neuronal MAP-2 and NeuN while nuclear DNA was stained with H33342. Neuronal survival was assessed using fluorescence microscopy and cell counting as described in Methods. Values are mean ± s.e.m.; n = 2 independent experiments with 4–7 replicates and an average of 5,300 cells counted per condition; ***p < 0.001, **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test.
Figure 3
Figure 3. IFNβ stimulates expression of interferon-stimulated genes (ISG) in mixed neuronal-glial cerebrocortical cell cultures.
Mixed neuronal-glial cerebrocortical cultures from WT or IFNAR1KO mice were incubated with IFNβ in increasing doses (from 500 to 5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h. Total RNA was extracted from cell lysates, analyzed by qRT-PCR and normalized to GAPDH expression levels. (a–d) RNA expression is shown as fold change (FC) in relation to vehicle treated controls which were defined as baseline activity. (e–h) Time course for protein expression measured in cell-free supernatants for CCL3, CCL4, CCL5 and CXCL10 using a commercially available multiplex assay as described in Methods. Baseline protein expression in vehicle treated cell cultures is represented as 0 h time point. Values are mean ± s.e.m.; n = 3–5 independent experiments per ISG; ***p < 0.001, **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test. For clarity, the significance is only indicated for differences between treatments and baseline within each experimental group.
Figure 4
Figure 4. Neuroprotection by IFNβ against HIVgp120 toxicity requires IFNAR1 and CCL4.
(a) Mixed neuronal-glial cerebrocortical cultures from WT mice were simultaneously exposed for 3 days to HIV gp120BaL (200 pM) and mouse IFNβ (5,000 U/ml) in the presence and absence of neutralizing antibodies against CCL3, CCL4, CCL5, IFNγ or CXCL10. IFNγ antibody was used as control for neutralizing antibodies since this protein was undetectable in cerebrocortical cell cultures. (b) Mouse cerebrocortical cultures from IFNAR1KO mice were stimulated with gp120BaL for 24 h the presence or absence of mouse IFNβ (5,000 U/ml) or BSA/PBS control. (c) Cerebrocortical cell cultures from WT mice were simultaneously exposed for 3 days to HIV gp120BaL in the presence and absence of murine CCL4 (2 or 20 nM). Neuronal survival was assessed by immunofluorescence microscopy and counting of MAP-2/NeuN double-positive neurons. Values are mean ± s.e.m.; n = 3–5 independent experiments with 3–7 replicates and an average of 9,000 (IFNAR1KO) or 5,700 (WT) cells counted per condition; **p < 0.01, ***p < 0.001 by ANOVA with Fisher’s PLSD post hoc test.
Figure 5
Figure 5. Intranasally administered IFNβ prevents neuronal damage in HIVgp120tg mice.
Three to four month-old HIVgp120tg and WT littermate male mice received intranasal recombinant murine IFNβ (50,000 U/25 g bodyweight) or vehicle once a week for four weeks and afterwards, the brains were assessed for neuronal damage and glial activation. Representative images of frontal cerebral cortex (a) and hippocampus (b) immunolabeled for neuronal synaptophysin (SYP; cortex layer III, hippocampus CA1) deconvolution microscopy; scale bar, 40 μm) and MAP-2 (cortex layer III (area between dashed lines) and hippocampus) fluorescence microscopy; scale bar, 100 μm), astrocytic GFAP (scale bar, 100 μm) and microglial Iba1 (red, DNA in blue, scale bar, 100 μm). (c–f) Quantification of microscopy data obtained in frontal cortex and hippocampus of sagittal brain sections of four to five month-old HIVgp120tg mice and controls treated with IFNβ or vehicle: (c) SYP in percent positive neuropil, (d) MAP-2 immunoreactivity as sum of fluorescence intensity (SFLI; arbitrary units); (e) fluorescence signal for astrocytic GFAP; (f) quantification of Iba1+ microglia (counts/microscopic field). Values are mean ± s.e.m.; ***p < 0.001, **p < 0.01, *p < 0.05; ANOVA and Fisher’s PLSD post hoc test; n = 4–5 animals per group/genotype.
Figure 6
Figure 6. Intranasal IFNβ treatment triggers expression of ISG in the brain.
RNA was purified from one brain hemisphere each of 4–5 month-old HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle and analyzed by qRT-PCR for fold-change (FC) in ISG expression. Significant changes in gene expression were observed between IFNβ and vehicle treatment groups in WT brains (a) for CCL4, and in gp120tg brains (b) for CCL4, CXCL11 and IRF3. Expression of transgenic HIVgp120 was not affected by IFNβ (c). Values are mean ± s.e.m.; n = 4–5 animals per group/genotype; *p < 0.05, student’s t-test.
Figure 7
Figure 7. Localization of CCL4 in vivo in astrocytes and neurons.
Sagittal brains sections of HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle (veh) were immunolabeled for CCL4, neuronal MAP-2 or astrocytic GFAP. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was labeled with Hoechst (H) 33342. The fluorescence-labeled brain sections were analyzed using confocal laser-scanning microscopy. Representative images of cortex layer III are shown; scale bar, 50 μm.
Figure 8
Figure 8. Interaction of IFNβ with neurons and astrocytes suffices to protect against neurotoxicity of HIVgp120-stimulated macrophages.
(a) Cerebrocortical cultures from mice were prepared to either contain microglia, neurons and astrocytes (M + N + A) or were depleted of microglia (N + A) or neurons and microglia (A). Complete and depleted cell cultures were incubated with mIFNβ (5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h and concentrations of CCL4 were measured in cell-free supernatants using a commercially available multiplex assay as described in Methods. Maximum concentrations were reached in samples of 12 to 24 h mIFNβ exposure and compared to vehicle-treated, baseline samples. Values are mean ± s.e.m.; n = 3 independent experiments; *p < 0.05, student’s t-test. (b) Microglia-depleted rat cerebrocortical cultures were exposed for 24 h to 50% cell-free conditioned media (CM) from human MDM in the presence or absence of human IFNβ (5,000 U/ml). MDM were previously stimulated for 24 h with HIV-1 gp120BaL (MDM gp120 CM) or vehicle (MDM CM). Following the incubation the cells were fixed and permeabilized. Neurons were immunolabeled for neuronal MAP-2 and NeuN and nuclear DNA was stained with H33342. Neuronal survival was assessed using fluorescence microscopy and cell counting as described in Methods. Values are mean ± s.e.m.; n = 2 independent experiments, with 4–8 replicates and an average of 4,000 cells counted per condition; **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test.

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