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Role of IFN-γ and LPS on neuron/glial co-cultures infected by Neospora caninum.

De Jesus EE, Santos AB, Ribeiro CS, Pinheiro AM, Freire SM, El-Bachá RS, Costa SL, de Fatima Dias Costa M - Front Cell Neurosci (2014)

Bottom Line: Infection or LPS treatment did not change NO production.Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%).The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Neuroquímica e Biologia Celular, Instituto de Ciências da Saúde, Universidade Federal da Bahia-UFBA Salvador, Brazil.

ABSTRACT
Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide (NO) production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 mg/mL of LPS on infected rat neuron/glial co-cultures. After 72 h of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not), meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%). The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in central nervous system (CNS).

No MeSH data available.


Related in: MedlinePlus

Immunodetection (immunoperoxidase) of tubulin βIII of rat neuron/glial cells co-culture treated with 300 IU/mL of IFN-γ and 1 mg/mL of LPS and infected with Neospora caninum tachyzoites (ratio cell:parasite 1:1), 72 h post infection. Control cells maintained in fresh medium exhibit a basal neurite outgrowth (arrow) (A); a drastic impairment of neurite (arrowhead) was observed in N. caninum infected culture (B) and IFN-γ treated cells (C); neurite length (arrow) in IFN-γ/infected cultures (D); LPS/uninfected and LPS/infected cells did not exhibit changes in neurite lenght (E,F). Scale bar = 50 µm. (G) Measurement and statistical analysis of neurite length (percentage of control) and its respective standard deviation compared to untreated/uninfected cultures (considered as 100%) in three samples in three independent experiments. “a” represents a significant statistical difference between the same treatment group; “b” represents a significant statistical difference when compared to untreated/uninfected control cultures; “c” represents a significant statistical difference when compared to untreated/infected cultures (Two-way ANOVA/Tukey’s Multiple Comparison Test—p < 0.05).
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Figure 5: Immunodetection (immunoperoxidase) of tubulin βIII of rat neuron/glial cells co-culture treated with 300 IU/mL of IFN-γ and 1 mg/mL of LPS and infected with Neospora caninum tachyzoites (ratio cell:parasite 1:1), 72 h post infection. Control cells maintained in fresh medium exhibit a basal neurite outgrowth (arrow) (A); a drastic impairment of neurite (arrowhead) was observed in N. caninum infected culture (B) and IFN-γ treated cells (C); neurite length (arrow) in IFN-γ/infected cultures (D); LPS/uninfected and LPS/infected cells did not exhibit changes in neurite lenght (E,F). Scale bar = 50 µm. (G) Measurement and statistical analysis of neurite length (percentage of control) and its respective standard deviation compared to untreated/uninfected cultures (considered as 100%) in three samples in three independent experiments. “a” represents a significant statistical difference between the same treatment group; “b” represents a significant statistical difference when compared to untreated/uninfected control cultures; “c” represents a significant statistical difference when compared to untreated/infected cultures (Two-way ANOVA/Tukey’s Multiple Comparison Test—p < 0.05).

Mentions: In this study, neurite outgrowth length was used as a parameter to evaluate the neuronal ability to maintain the dynamics of the tubulin and actin cytoskeletons (Figure 5), which is essential for the establishment of synapses. The basal neurite length under control conditions was considered as 100%. Untreated/infected neuron/glial co-cultures showed a drastic impairment of neurite outgrowth (reduction of 51.47%; p < 0.001), which can represent a possible deleterious effect of parasite infection. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%).


Role of IFN-γ and LPS on neuron/glial co-cultures infected by Neospora caninum.

De Jesus EE, Santos AB, Ribeiro CS, Pinheiro AM, Freire SM, El-Bachá RS, Costa SL, de Fatima Dias Costa M - Front Cell Neurosci (2014)

Immunodetection (immunoperoxidase) of tubulin βIII of rat neuron/glial cells co-culture treated with 300 IU/mL of IFN-γ and 1 mg/mL of LPS and infected with Neospora caninum tachyzoites (ratio cell:parasite 1:1), 72 h post infection. Control cells maintained in fresh medium exhibit a basal neurite outgrowth (arrow) (A); a drastic impairment of neurite (arrowhead) was observed in N. caninum infected culture (B) and IFN-γ treated cells (C); neurite length (arrow) in IFN-γ/infected cultures (D); LPS/uninfected and LPS/infected cells did not exhibit changes in neurite lenght (E,F). Scale bar = 50 µm. (G) Measurement and statistical analysis of neurite length (percentage of control) and its respective standard deviation compared to untreated/uninfected cultures (considered as 100%) in three samples in three independent experiments. “a” represents a significant statistical difference between the same treatment group; “b” represents a significant statistical difference when compared to untreated/uninfected control cultures; “c” represents a significant statistical difference when compared to untreated/infected cultures (Two-way ANOVA/Tukey’s Multiple Comparison Test—p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Immunodetection (immunoperoxidase) of tubulin βIII of rat neuron/glial cells co-culture treated with 300 IU/mL of IFN-γ and 1 mg/mL of LPS and infected with Neospora caninum tachyzoites (ratio cell:parasite 1:1), 72 h post infection. Control cells maintained in fresh medium exhibit a basal neurite outgrowth (arrow) (A); a drastic impairment of neurite (arrowhead) was observed in N. caninum infected culture (B) and IFN-γ treated cells (C); neurite length (arrow) in IFN-γ/infected cultures (D); LPS/uninfected and LPS/infected cells did not exhibit changes in neurite lenght (E,F). Scale bar = 50 µm. (G) Measurement and statistical analysis of neurite length (percentage of control) and its respective standard deviation compared to untreated/uninfected cultures (considered as 100%) in three samples in three independent experiments. “a” represents a significant statistical difference between the same treatment group; “b” represents a significant statistical difference when compared to untreated/uninfected control cultures; “c” represents a significant statistical difference when compared to untreated/infected cultures (Two-way ANOVA/Tukey’s Multiple Comparison Test—p < 0.05).
Mentions: In this study, neurite outgrowth length was used as a parameter to evaluate the neuronal ability to maintain the dynamics of the tubulin and actin cytoskeletons (Figure 5), which is essential for the establishment of synapses. The basal neurite length under control conditions was considered as 100%. Untreated/infected neuron/glial co-cultures showed a drastic impairment of neurite outgrowth (reduction of 51.47%; p < 0.001), which can represent a possible deleterious effect of parasite infection. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%).

Bottom Line: Infection or LPS treatment did not change NO production.Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%).The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Neuroquímica e Biologia Celular, Instituto de Ciências da Saúde, Universidade Federal da Bahia-UFBA Salvador, Brazil.

ABSTRACT
Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide (NO) production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 mg/mL of LPS on infected rat neuron/glial co-cultures. After 72 h of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not), meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%). The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in central nervous system (CNS).

No MeSH data available.


Related in: MedlinePlus