CXCR3 activation by lentivirus infection suppresses neuronal autophagy: neuroprotective effects of antiretroviral therapy

Abstract

Previous studies have implicated CXCL12 in the neuropathogenesis of HIV infection. Proteolysis of CXCL12 generates a neurotoxic molecule, CXCL12(5-67), which engages and activates CXCR3, in addition to exhibiting increased expression in the brains of patients with HIV-associated dementia (HAD). Herein, we investigated CXCR3-mediated neuronal injury, particularly, its contribution to autophagy suppression and the concomitant effects of antiretroviral therapy using human brain samples and models of HIV neuropathogenesis. Neurons in the brains of HAD patients and feline immunodeficiency virus (FIV)-infected animals, as well as cultured human neurons, expressed CXCR3, which was modulated in a ligand-specific manner. Exposure of human neurons to CXCL12(5-67) caused a reduction in the autophagy-associated molecule LC3 (P<0.05) and neuronal survival (P<0.05), which recapitulated findings in FIV- and HIV-infected brains (P<0.05). Oral didanosine (ddI) treatment of FIV-infected animals reduced neurobehavioral abnormalities in conjunction with diminished plasma viral load (P<0.05). F4/80 transcript abundance and CXCL12(5-67) immunoreactivity were reduced with restored neuronal LC3 expression in the brains of FIV-infected animals after ddI treatment (P<0.05). ddI treatment also prevented microglial activation and depletion of synaptic proteins in the cortex of FIV-infected animals (P<0.05). These findings indicate that the beneficial effects of ddI might be a consequence of a reduced systemic viral burden and concurrent leukocyte activation, leading to diminished neuroinflammation with preservation of neuronal autophagy by regulating CXCR3 activation.

Figure 1.

CXCR3 isoforms and autophagy regulation in HIV-infected human brains. Neuronal nuclear maker NeuN was readily detected in non-HIV-infected (HIV⁻) brain tissue (A), compared with fewer immunopositive neurons in the HIV-infected (HIV⁺) brain (B). CXCR3 immunoreactivity was detected in neurons of brains from HIV⁻ persons (C), but there was more abundant CXCR3 immunoreactivity in HIV⁺ samples (D), including basal ganglia (D, inset). LC3-immunoreactive cells were evident in the HIV⁻ brain (E), but fewer cells were observed in the HIV⁺ brain (F), although LC3 immunoreactivity (brown; F, inset) colocalized with NeuN (blue; F, inset). CXCR3 immunoreactivity in human brain lysates in Western blot revealed two bands (G), showing an increase in intensity confirmed by quantification (H). CXCR3A transcript expression was higher in the brains of patients diagnosed with HAD compared with HIV⁻ brains or ND; CXCR3B expression was higher in both demented and nondemented HIV⁺ brains compared with HIV⁻ brains (I). LC3 immunoreactivity of brain lysates on Western blot also showed two bands (J), although the bottom (lipidated) band was suppressed in HAD brains (K). p62 transcript levels showed a trend toward increased expression in HIV⁺ brains (L). *P < 0.05; ANOVA. Original view: ×400 (A–D; insets); ×200 (E, F).

Figure 2.

CXCR3 isoforms are differentially regulated by inflammatory mediators. A) Expression of CXCR3A and CXCR3B in HFN treated with 10 or 20 ng/ml of IL-1β. B–D) Relative fold change (RFC) in transcript abundance of CXCR3A and CXCR3B in human fetal neuronal cultures treated with 10 or 100 nM of either CXCL12(5-67) (D), CXCL10 (C), or CXCL4 (B). E) Percentage of cell surface CXCR3 on neuronal (LAN-2) cells, expressed as ratio of CXCR3 immunoreactivity in nonpermeabilized to permeabilized cells, measured by In Cell Western blot, showed a concentration-dependent decrease after CXCL12(5-67) exposure for 8 h. F) Western blots revealed a concentration-dependent increase in β-arrestin-1 immunoreactivity in neuronal cells on exposure to 10, 100, or 1000 nM of CXCL12(5-67) compared with control; exposure to full-length CXCL12 showed only a modest increase in β-arrestin-1 abundance. As controls, Western blots did not show any significant modulation of CXCR3 or β-actin after the same treatment. *P < 0.05, **P < 0.01 vs. no treatment; ANOVA.

Figure 3.

Neurobehavioral performance, viral burden, and neuroinflammation in FIV infection. A) FIV-infected animals (FIV+) exhibit greater neurobehavioral deficits than mock-infected controls (FIV−), while ddI treatment reversed neurobehavioral deficits in FIV-infected animals (FIV+/ddI) and had no effects on mock-infected animals. B) ddI treatment suppressed viral load in plasma of FIV-infected animals. C) Glycoprotein, F4/80, expressed in activated myeloid cells showed increased transcript abundance in FIV-infected animals, which was reversed by ddI treatment. D) CXCL12 transcript levels were higher in FIV-infected animals but were not reduced by ddI treatment. E) MMP-2 transcripts were unregulated in FIV-infected animals but were suppressed with ddI treatment. F) GFAP transcriptional activity was not affected by FIV infection or ddI treatment. *P < 0.05; Dunnet multiple comparison test.

Figure 4.

Brain expression of CXCL12, MMP-2, and CXCR3 in FIV infection. A) Immunoblotting showed increased full-length (1-67) and cleaved (5-67) CXCL12 in brains of FIV⁻ compared with mock-infected animals. B) CXCL12, CXCL12(5-67), MMP-2, and CXCR3 immunoreactivities were increased in brains of FIV⁺ animals but were suppressed by concurrent ddI treatment. C) MMP-2 immunoreactivity (IR) showed minimal induction in FIV⁺ animals with and without ddI treatment. D, E) CXCL12(1-67) (D) and CXCL12(5-67) (E) were induced by FIV infection, but these changes were reversed by ddI treatment. F) CXCR3 showed minimal induction of both immunoreactive bands in FIV infection and was not affected by ddI treatment. *P < 0.05; Dunnet multiple comparison test.

Figure 5.

Suppression of autophagy in neurons by CXCL12(5-67) and FIV infection. A) CXCL12(5-67) reduced human neuronal viability in terms of β-tubulin immunoreactivity (i) and cell counts/hpf (ii). B) CXCL12(5-67) exposure reduced LC3 immunoreactivity (i) by >50% in human primary neurons (ii). C) LC3 immunoreactivity, including its lipidated form, was detectable in human neuronal (LAN-2) cells, but CXCL12(5-67) exposure reduced LC3 immunoreactivity, which was blocked by concomitant CXCL11(5-73) treatment (i), although CXCL11(5-73) alone did not affect LC3 expression (ii). D) LC3 immunoreactivity (i) was suppressed in the brains of FIV-infected animals but was rescued by ddI treatment (ii). E) The autophagy-associated gene atg5 was suppressed in brains of FIV⁺ animals, although this change was partially reversed by ddI treatment. *P < 0.05; Dunnet multiple comparison test.

Figure 6.

Neuropathological changes associated with FIV infection and ddI treatment. Iba-1-immunoreactive microglia were detected in brains of mock-infected FIV⁻ animals (A) but were more abundant and hypertrophied in FIV⁺ brains (B), and ddI treatment reversed this activation of microglia effect (C). NeuN-immunopositive neurons were detected in the parietal cortex of mock-infected controls (D), but were reduced in frequency in FIV⁺ animals (E), although ddI treatment appeared to protect neurons (F). CXCL12(5-67)-positive cells were not detected in mock-infected controls (G); in contrast CXCL12 immunoreactivity was observed in FIV-infected brains (H) but minimally detected in FIV-infected and ddI-treated animals (I). Inset shows colocalization of CXCL12(5-67) and NeuN in neurons. CXCR3 immunopositive cells resembling neurons were detected in mock-infected (J), FIV⁺ (K), and FIV⁺ animals receiving ddI (L). LC3-immunopositive cells colabeled with NeuN were readily found in mock-infected brains (M), but were reduced in number in FIV⁺ animals (N); ddI treatment appeared to reverse LC3 immunoreactivity in FIV infection (O). Synaptic protein VAChT was detected in brains of all groups (P). However, VAChT was reduced in FIV infection, but ddI treatment prevented the suppression of this synaptic protein (Q). Original view: ×630. *P < 0.05; Dunnet multiple comparison test.

Figure 7.

Suppression of neuronal autophagy and its regulation by antiretroviral therapy. ddI suppressed viral burden in the peripheral circulation, diminishing leukocyte activation and CNS entry with an ensuing reduction in MMP-2 production and its actions, thereby preventing the induction of CXCL12(5-12) with its adverse effects on neuronal autophagy and survival.