Interleukin-10 Inhibits Neuroinflammation And Dopaminergic Neuronal Apoptosis

Objectives: IL-10 is the most important and best characterized anti-inflammatory cytokine. It plays an important role in the regulation of inflammation and immune response. Recently, the neuroinflammation has been reported to participate in pathogenesis of Parkinson’s disease(PD). In the central nervous system(CNS), expression of IL-10 or IL-10 R has been described in neurons, microglia, astrocytes or oligodendrocytes, in both basal conditions and after injury, indicating a potentially broad range of actions of this cytokine in the CNS. In addition, IL-10 may also play a role of anti-apoptosis in neurons, and we speculate that IL-10 can prevent chronic inflammation related diseases by its anti-inflammatory and anti-apoptotic effects, such as PD. Herein, we explore the effects of IL-10 on neuroinflammatory processes and dopaminergic neurons loss, reveal the underlying mechanisms of IL-10, and provide a new cue for IL-10 alleviation of PD neurodegeneration.Methods: 1.Cell culture:(1) Primary ventral mesencephalic(VM) Cultures:Fetuses obtained from pregnant Sprague-Dawley rats on the 14 th ± 0.5 gestational days were used for preparation of ventral mesencephalic neuron-glial cultures. Seven-day-old cultures were used for treatments.(2) Primary VM neuron-enriched Cultures:VM cultures were prepared from the VM tissues of embryonic Day 14(E14) ± 0.5 Sprague-Dawley rats. On the second day after initial seeding, cytosine β-D-arabinofuranoside(10 μM) was added to suppress glial proliferation. Seven-day-old cultures were used for treatments.(3) Primary neuron-microglia co-cultures:Primary microglias were isolated from cerebral cortices of newborn Sprague-Dawley rats. 12-14 days later, microglia were harvested and added to neuron-enriched cultures. One day later, the reconstituted cell cultures were used for treatments.(4) Primary neuron-astrocyte co-cultures:Cerebral cortices were processed into glial cell cultures. 12-14 days later, the remnant cells after the separation of microglia were consecutive passaged at least four times to obtain primary astrocytes. Neuron-astrocyte co-cultures were prepared by co-culturing fetal VM neurons with the nearly pure astrocytes on the sixth day after the fetal VM neurons from E14 ± 0.5 rats were cultured. One day later, the neuron-astrocyte co-cultures were used for treatments. 2. In the four cultures, the effects of IL-10 on neuroinflammation and dopaminergic neurons loss induced by LPS were evaluated: The cultures were pretreated with recombinant rat IL-10(15, 50, or 150 ng/m L) for 1h and then the neurotoxin LPS(50 ng/m L) was added, which were incubated for 8h or 24 h. The number of TH+Neu N+ dopaminergic neurons and TH-Neu N+ non-dopaminergic neurons was counted by immunocytochemical staining. The expression of the inflammatory mediators(i NOS, COX-2, IL-1β and TNF-α) and the neurotrophic factors(BDNF, IGF-1, and GDNF) was measured by real-time PCR and Western blot. The concentration of IL-1β, TNF-α or IGF-1 was detected by ELISA. In addition, the concentration of NO and H2O2 was tested by chemical colorimetry in the primary VM cultures. Double-immunofluorescent staining for TLR-4 with Neu N was detected in the primary VM neuron-enriched cultures, the protein expression of activated caspase-3 and activated caspase-9 was tested by Western blot, and the apoptotic cells were detected by TUNEL staining. 3.In the primary VM cultures, the effects of IL-10 on neuroinflammation and dopaminergic neurons loss induced by MPP+ were detected: The cultures were pretreated with IL-10(15 or 50 ng/m L) for 1h and then MPP+(5μM) was added, which were incubated for 24 h. The number of TH-immunreactive dopaminergic neurons was counted by immunocytochemical staining. The protein expression of the inflammatory mediators(i NOS, COX-2, IL-1β and TNF-α) and the neurotrophic factors(BDNF, IGF-1, and GDNF) was measured by Western blot. The concentration of IL-1β, TNF-α and IGF-1 was detected by ELISA.IL-10(15 or 50ng/m L) were added into the primary VM neuron-enriched cultures 1 h before MPP+(5 μM) was applied, which were incubated for 8 h or 24 h. The number of TH-immunreactive dopaminergic neurons was tested by immunocytochemical staining. The expression of activated caspase-3 and activated caspase-9 was measured by Western blot. Double-immunofluorescent staining for TNF-α with TH was detected in the cultures. The level of TNF-α was measured by Western blot and ELISA. The neurons, neutralized with TNF-α antiboby(1 μg/m L) for 2 h or not, were treated with IL-10 for 1 h followed by MPP+(5 μM) application. They were incubated for 8 h or 24 h. The number of dopaminergic neurons was counted by immunocytochemical staining. The expression of activated caspase-3 and activated caspase-9 with various treatments were evaluated by Western blot. 5.In the primary VM neuron-enriched cultures, the alleviative effects of IL-10 via IL-10Rα and JAK-STAT3 signaling pathway on LPS-induced neurotoxicity were determined: Double-immunofluorescent staining for IL-10Rα with Neu N was detected in the VM neuron-enriched cultures. After Silencing of IL-10Rα gene in neurons or application of JAK inhibitor(0.5, or 1 μM), the neurons were treated with IL-10(50 ng/m L) for 1h and then LPS(50 ng/m L) was added to the cultures, which were incubated for 8 h or 24 h. Immunocytochemical staining was employed to detect the number of dopaminergic and non-dopaminergic neurons, respectively; Western blot was applied to estimate the expression of COX-2, TNF-α, BDNF, GDNF, activated caspase-3, and activated caspase-9; ELISA was used to examine the concentration of TNF-α. In the case of pretreatment with JAK inhibitors, the neuron apoptosis was detected by TUNEL staining, and the expression of phosphorylated Stat3 with various treatments was evaluated by Western blot. 4. In the primary VM neuron-enriched cultures, the effects of IL-10 on neuroinflammation and dopaminergic neurons loss induced by MPP+ were determined:Results: 1. IL-10 attenuated neuroinflammation and dopaminergic neurons loss induced by LPS in the primary VM cultures: In primary VM cultures, LPS induced a decrease in the number of TH+Neu N+ and TH-Neu N+ cells, upregulation of pro-inflammatory mediators(i NOS, COX-2, IL-1β and TNF-α), and downregulation of neurotrophic factor(BDNF). But IGF-1, also a neurotrophic factor, was upregulated by LPS. In addition, LPS induced a rapid accumulation of NO and H2O2 in the supernatant of VM cultures. IL-10 attenuated the toxic effects induced by LPS. The expression of GDNF did not change obviously. 2. IL-10 attenuated neuroinflammation and dopaminergic neurons loss induced by MPP+ in the primary VM cultures: MPP+ induced TH+ neuron loss in primary VM cultures, which was blocked by IL-10 administration. In addition, IL-10 was effective in attenuating cellular expression of a host of inflammatory mediators(i NOS, COX-2, IL-1β, and TNF-α) in response to toxicant and counteracted MPP+-induced reduction in the expression of all neurotrophic factors(BDNF, IGF-1, and GDNF). 3. IL-10 attenuated neuroinflammation and dopaminergic neurons loss induced by LPS in the primary neuron–microglia co-cultures: LPS induced dopaminergic and non-dopaminergic neuronal loss, upregulation of pro-inflammatory mediators(i NOS, COX-2, IL-1β, and TNF-α), and downregulation of neurotrophic factors(BDNF, IGF-1 or GDNF). IL-10 offered significant protection against LPS-induced damage to TH+Neu N+ and TH-Neu N+ cells. IL-10 also inhibited both the increase in production of the pro-inflammatory mediators and the decrease in production of the neurotrophic factors induced by LPS. 4. IL-10 attenuated upregulated expression of inflammatory mediators induced by LPS in the primary neuron-astrocyte co-cultures: The LPS alone did not affect the number of TH+Neu N+ and TH-Neu N+ neurons in neuron-astrocyte co-cultures. Pro-inflammatory mediators(i NOS, COX-2, IL-1β, and TNF-α) still were upregulated by LPS, meanwhile, neurotrophic factors(BDNF, and IGF-1) were upregulated too(although IGF-1 protein expression was not detected). The expression of GDNF did not change obviously. IL-10 reduced the increase in production of the pro-inflammatory mediators, but did not significantly alter production of BDNF induced by LPS. 5. IL-10 attenuated MPP+-induced neuroinflammation and dopaminergic neurons loss in primary VM neuron-enriched cultures: We found that TNF-α was co-localized in TH+ dopaminergic neurons. MPP+ induced dopaminergic neuronal loss in primary VM neuron-enriched cultures, while IL-10 reduced neuronal loss induced by MPP+. MPP+ increased the level of TNF-α,actived caspase-3 and actived caspase-9, while IL-10 prevented the MPP+-induced protein expression changes. Inhibition of TNF-α suppressed MPP+-induced dopaminergic neuron apoptosis by declining the expression of pro-apoptotic proteins. 6. IL-10 attenuated LPS-induced neuroinflammation and dopaminergic neurons loss via IL-10Rα and JAK-STAT3 signaling pathway in the primary VM neuron-enriched cultures: We found that TLR-4 was co-localized with neurons. LPS enhanced the percentage of TUNEL-positive neurons and decreased the number of dopaminergic and non-dopaminergic neurons in primary VM neuron-enriched cultures. LPS upregulated expression of TNF-α and pro-apoptosis protein, but downregulated expression of BDNF and the phosphorylation level of Stat3. The combined treatment with IL-10 and LPS increased both TH+Neu N+ and TH-Neu N+ cell number and reduced the production of TNF-α, pro-apoptotic proteins and the percentage of TUNEL-positive cells compared with the LPS toxicity. IL-10 also increased the production of BDNF and the phosphorylation level of Stat3, but did not significantly affect the level of COX-2. Other pro-inflammatory mediators(i NOS and IL-1β) and neurotrophic factor(IGF-1) were not detected for their expression. We also found that IL-10Rα was co-localized with neurons. But IL-10 could not inhibit neurons loss when we knockdown the IL-10Rα gene in neurons. In addition, Silencing of IL-10Rα gene in neurons or JAK inhibitor weakened the role of IL-10 in prevention of neurotoxicity of LPS. The protein expression of COX-2 and GDNF did not change obviously.Conclusions: Collectively, our findings suggest that IL-10 exerts neuroprotective property by an extensive action including direct on neurons and indirect via inhibiting glial over-activation. IL-10 attenuates neuronal loss in vitro, which is mediated through altering the expression of inflammatory mediators and neurotrophic factors initiated by glial cells and neurons. Moreover, we provide evidence that the IL-10Rα receptor and downstream JAK-STAT3 signaling pathway are involved in the neuroprotective actions of IL-10. Altogether, these results may participate in the understanding of IL-10’s function in neurodegenerative diseases.