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HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity.

Mangino G, Famiglietti M, Capone C, Veroni C, Percario ZA, Leone S, Fiorucci G, Lülf S, Romeo G, Agresti C, Persichini T, Geyer M, Affabris E - PLoS ONE (2015)

Bottom Line: We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef.All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein.These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model.

View Article: PubMed Central - PubMed

Affiliation: Department of Science, University Roma Tre, Rome, Italy; Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy.

ABSTRACT

Background: The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors.

Methodology/principal findings: We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells.

Conclusions: These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.

No MeSH data available.


Related in: MedlinePlus

Nef treatment of microglial cells induces iNOS expression.BV-2 cells (A) or purified primary murine microglial cells (B) were treated for the indicated time with myr+NefSF2 (100 ng/ml in A, 200 ng/ml in B, closed circles). Total cellular RNAs were isolated and real time RT-PCR analysis was performed as reported in the materials and methods section. Results were expressed using the 2-ΔΔCT method using basal mRNA level in untreated cells (Ctr, open circles) at T = 0 as a calibrator and GAPDH level as an internal loading control. (C, D) BV-2 cells were treated for 24 h with the indicated amounts of myr+NefSF2, heat inactivated myr+NefSF2 (Inactiv. recNef), LPS or heat treated LPS. Total cell lysates were analyzed by Western Blot for iNOS (C, upper panel) levels and, as internal loading control, β-Tubulin expression (C, lower panel). (D) NO2- content in the supernatants was quantified using the Griess colorimetric assay as reported in the materials and methods section. Dark gray bars: native myr+Nef or LPS. Light gray bars: heat pre-treated (Inactiv.) myr+Nef or heat pre-treated LPS. Results from one of five independent experiments are shown. (E) Cell phenotyping by flow cytometry of human monocytes and M1 macrophages obtained as described in materials and methods. According to [77], human monocytes were CD14+/CD163+/CD86+ whereas M1 macrophages were CD14-/CD163-/CD86bright. (F) M1 human macrophages were left untreated or treated for 24, 48 and 72 h with IFNγ, 100 ng/ml wild type myr+NefSF2 plus IFNγ, heat pre-treated myr+NefSF2 and IFNγ or 100 ng/ml LPS plus IFNγ. Total cellular extracts were analyzed by Western Blot to evaluate iNOS expression using specific antibodies as described in materials and methods. β-tubulin expression as internal loading control. Blots are representative of two independent experiments.
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pone.0130189.g002: Nef treatment of microglial cells induces iNOS expression.BV-2 cells (A) or purified primary murine microglial cells (B) were treated for the indicated time with myr+NefSF2 (100 ng/ml in A, 200 ng/ml in B, closed circles). Total cellular RNAs were isolated and real time RT-PCR analysis was performed as reported in the materials and methods section. Results were expressed using the 2-ΔΔCT method using basal mRNA level in untreated cells (Ctr, open circles) at T = 0 as a calibrator and GAPDH level as an internal loading control. (C, D) BV-2 cells were treated for 24 h with the indicated amounts of myr+NefSF2, heat inactivated myr+NefSF2 (Inactiv. recNef), LPS or heat treated LPS. Total cell lysates were analyzed by Western Blot for iNOS (C, upper panel) levels and, as internal loading control, β-Tubulin expression (C, lower panel). (D) NO2- content in the supernatants was quantified using the Griess colorimetric assay as reported in the materials and methods section. Dark gray bars: native myr+Nef or LPS. Light gray bars: heat pre-treated (Inactiv.) myr+Nef or heat pre-treated LPS. Results from one of five independent experiments are shown. (E) Cell phenotyping by flow cytometry of human monocytes and M1 macrophages obtained as described in materials and methods. According to [77], human monocytes were CD14+/CD163+/CD86+ whereas M1 macrophages were CD14-/CD163-/CD86bright. (F) M1 human macrophages were left untreated or treated for 24, 48 and 72 h with IFNγ, 100 ng/ml wild type myr+NefSF2 plus IFNγ, heat pre-treated myr+NefSF2 and IFNγ or 100 ng/ml LPS plus IFNγ. Total cellular extracts were analyzed by Western Blot to evaluate iNOS expression using specific antibodies as described in materials and methods. β-tubulin expression as internal loading control. Blots are representative of two independent experiments.

Mentions: Based on the above results, we asked whether extracellular Nef was able to regulate iNOS expression. First, BV-2 cells were exposed to myr+NefSF2 and iNOS mRNA levels were evaluated by Real Time RT-PCR from 1 to 24 h post-treatment. As shown in Fig 2A, iNOS mRNA expression reached a peak at 6 h post-treatment, declined thereafter and increased again at 16 and 24 h, similarly to the kinetics of iNOS mRNA induction in cytokines-stimulated DLD-1 cells [44]. Induction of iNOS mRNA expression was also confirmed in primary murine microglial cells (Fig 2B). To test whether iNOS expression and NO2- production were both affected, BV-2 cells were treated for 24 h with 100 to 500 ng/ml myr+NefSF2. iNOS protein levels were measured in total cellular extract, whereas quantification of NO2- released into the supernatants was performed using the Griess reagent. As depicted in Fig 2C and 2D, iNOS expression and NO2- release were induced in a dose-dependent manner by myr+Nef treatment of microglial cells. The effect required the integrity of the protein, as heat-denaturated Nef barely induced iNOS synthesis and NO2- release. As a positive control, we evaluated NO2- production induced by LPS which is not sensitive to heat inactivation. To verify iNOS induction in a human context, we polarized monocytes into M1 inflammatory macrophages by cultivating them for 6 days in the presence of GM-CSF (Fig 2E). M1 macrophages were then treated with myr+Nef in the presence of IFNγ for 24, 48 or 72 h. We were unable to detect nitrite accumulation in the supernatants of Nef-treated M1 macrophages, due to the well known low NO2-/NO3- ratio production in human cells [45], nevertheless we found that iNOS was slightly induced in cells treated for 48 h with myr+Nef plus IFNγ compared to IFNγ treatment alone (Fig 2F and S1 File). Stimulation with LPS plus IFNγ, used as a positive control, induced iNOS at 24 and 48 h declining thereafter at 72 h (Fig 2F).


HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity.

Mangino G, Famiglietti M, Capone C, Veroni C, Percario ZA, Leone S, Fiorucci G, Lülf S, Romeo G, Agresti C, Persichini T, Geyer M, Affabris E - PLoS ONE (2015)

Nef treatment of microglial cells induces iNOS expression.BV-2 cells (A) or purified primary murine microglial cells (B) were treated for the indicated time with myr+NefSF2 (100 ng/ml in A, 200 ng/ml in B, closed circles). Total cellular RNAs were isolated and real time RT-PCR analysis was performed as reported in the materials and methods section. Results were expressed using the 2-ΔΔCT method using basal mRNA level in untreated cells (Ctr, open circles) at T = 0 as a calibrator and GAPDH level as an internal loading control. (C, D) BV-2 cells were treated for 24 h with the indicated amounts of myr+NefSF2, heat inactivated myr+NefSF2 (Inactiv. recNef), LPS or heat treated LPS. Total cell lysates were analyzed by Western Blot for iNOS (C, upper panel) levels and, as internal loading control, β-Tubulin expression (C, lower panel). (D) NO2- content in the supernatants was quantified using the Griess colorimetric assay as reported in the materials and methods section. Dark gray bars: native myr+Nef or LPS. Light gray bars: heat pre-treated (Inactiv.) myr+Nef or heat pre-treated LPS. Results from one of five independent experiments are shown. (E) Cell phenotyping by flow cytometry of human monocytes and M1 macrophages obtained as described in materials and methods. According to [77], human monocytes were CD14+/CD163+/CD86+ whereas M1 macrophages were CD14-/CD163-/CD86bright. (F) M1 human macrophages were left untreated or treated for 24, 48 and 72 h with IFNγ, 100 ng/ml wild type myr+NefSF2 plus IFNγ, heat pre-treated myr+NefSF2 and IFNγ or 100 ng/ml LPS plus IFNγ. Total cellular extracts were analyzed by Western Blot to evaluate iNOS expression using specific antibodies as described in materials and methods. β-tubulin expression as internal loading control. Blots are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4465743&req=5

pone.0130189.g002: Nef treatment of microglial cells induces iNOS expression.BV-2 cells (A) or purified primary murine microglial cells (B) were treated for the indicated time with myr+NefSF2 (100 ng/ml in A, 200 ng/ml in B, closed circles). Total cellular RNAs were isolated and real time RT-PCR analysis was performed as reported in the materials and methods section. Results were expressed using the 2-ΔΔCT method using basal mRNA level in untreated cells (Ctr, open circles) at T = 0 as a calibrator and GAPDH level as an internal loading control. (C, D) BV-2 cells were treated for 24 h with the indicated amounts of myr+NefSF2, heat inactivated myr+NefSF2 (Inactiv. recNef), LPS or heat treated LPS. Total cell lysates were analyzed by Western Blot for iNOS (C, upper panel) levels and, as internal loading control, β-Tubulin expression (C, lower panel). (D) NO2- content in the supernatants was quantified using the Griess colorimetric assay as reported in the materials and methods section. Dark gray bars: native myr+Nef or LPS. Light gray bars: heat pre-treated (Inactiv.) myr+Nef or heat pre-treated LPS. Results from one of five independent experiments are shown. (E) Cell phenotyping by flow cytometry of human monocytes and M1 macrophages obtained as described in materials and methods. According to [77], human monocytes were CD14+/CD163+/CD86+ whereas M1 macrophages were CD14-/CD163-/CD86bright. (F) M1 human macrophages were left untreated or treated for 24, 48 and 72 h with IFNγ, 100 ng/ml wild type myr+NefSF2 plus IFNγ, heat pre-treated myr+NefSF2 and IFNγ or 100 ng/ml LPS plus IFNγ. Total cellular extracts were analyzed by Western Blot to evaluate iNOS expression using specific antibodies as described in materials and methods. β-tubulin expression as internal loading control. Blots are representative of two independent experiments.
Mentions: Based on the above results, we asked whether extracellular Nef was able to regulate iNOS expression. First, BV-2 cells were exposed to myr+NefSF2 and iNOS mRNA levels were evaluated by Real Time RT-PCR from 1 to 24 h post-treatment. As shown in Fig 2A, iNOS mRNA expression reached a peak at 6 h post-treatment, declined thereafter and increased again at 16 and 24 h, similarly to the kinetics of iNOS mRNA induction in cytokines-stimulated DLD-1 cells [44]. Induction of iNOS mRNA expression was also confirmed in primary murine microglial cells (Fig 2B). To test whether iNOS expression and NO2- production were both affected, BV-2 cells were treated for 24 h with 100 to 500 ng/ml myr+NefSF2. iNOS protein levels were measured in total cellular extract, whereas quantification of NO2- released into the supernatants was performed using the Griess reagent. As depicted in Fig 2C and 2D, iNOS expression and NO2- release were induced in a dose-dependent manner by myr+Nef treatment of microglial cells. The effect required the integrity of the protein, as heat-denaturated Nef barely induced iNOS synthesis and NO2- release. As a positive control, we evaluated NO2- production induced by LPS which is not sensitive to heat inactivation. To verify iNOS induction in a human context, we polarized monocytes into M1 inflammatory macrophages by cultivating them for 6 days in the presence of GM-CSF (Fig 2E). M1 macrophages were then treated with myr+Nef in the presence of IFNγ for 24, 48 or 72 h. We were unable to detect nitrite accumulation in the supernatants of Nef-treated M1 macrophages, due to the well known low NO2-/NO3- ratio production in human cells [45], nevertheless we found that iNOS was slightly induced in cells treated for 48 h with myr+Nef plus IFNγ compared to IFNγ treatment alone (Fig 2F and S1 File). Stimulation with LPS plus IFNγ, used as a positive control, induced iNOS at 24 and 48 h declining thereafter at 72 h (Fig 2F).

Bottom Line: We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef.All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein.These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model.

View Article: PubMed Central - PubMed

Affiliation: Department of Science, University Roma Tre, Rome, Italy; Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy.

ABSTRACT

Background: The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors.

Methodology/principal findings: We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells.

Conclusions: These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.

No MeSH data available.


Related in: MedlinePlus