Dimethyl Fumarate, an Immune Modulator and Inducer of the Antioxidant Response, Suppresses HIV Replication and Macrophage-Mediated Neurotoxicity: A Novel Candidate for HIV Neuroprotection

DMF and MMF reduce HIV/MDM-mediated neurotoxicity. Rat cerebrocortical cultures were exposed to supernatant from HIV-infected macrophages that were treated with DMF (A) or MMF (B) at the indicated concentrations (1–30 μM) during the course of infection. Neuronal survival was assessed by MAP2 ELISA and expressed as a percentage of untreated (UT) cultures (n = 6). C, Representative images of rat cerebrocortical cultures immunofluorescently stained for MAP2 (red) and Hoechst 33324 (blue) following 24 h treatment with the indicated HIV/MDM supernatant. Scale bar, 50 μm. All statistical comparisons were made by one-way ANOVA plus Newman–Keuls post hoc testing. ***p < 0.001 versus vehicle.

DMF inhibits NF-κB nuclear translocation, DNA binding, and TNF-α production in human MDM. DMF (A) and MMF (B) inhibit the nuclear translocation of the NF-κB proteins RelB, p65, and p50 in human MDM in a dose-dependent manner. Cells were treated with DMF or MMF for 24 h, exposed to TNF-α (10 min), separated into cytoplasmic and nuclear fractions, and analyzed by Western blotting. Results of densitometry analysis are presented numerically under each panel as the ratio of NF-κB protein to poly(ADP-ribose) polymerase, a nuclear marker, and loading control. Blots are representative of four to six independent experiments, with each replicate performed on cell preparations from different donors. C, DMF inhibits nuclear NF-κB p50 binding to DNA in TNF-α–stimulated MDM, as assessed by EMSA. Results of densitometry analysis were normalized to vehicle. D, DMF inhibits the production of TNF-α in MDM stimulated with PHA (10 μg/ml). Values are expressed as percent TNF-α production relative to vehicle-treated cells (227 ± 11.9 pg/ml TNF-α in vehicle). Data are expressed as mean ± SEM and averaged from four different donors. E, TNF-α production in HIV/MDM is inhibited by DMF treatment. HIV/MDM were treated with DMF (1–30 μM) or 20 nM EFZ over the course of infection and culture supernatants from days 14 to 15 postinfection were assayed for TNF-α by ELISA. Values represent the mean ± SEM of data averaged from four different donors. All statistical comparisons were made by one-way ANOVA plus Newman–Keuls post hoc testing. *p < 0.05, **p < 0.01, ***p < 0.01 versus vehicle.

DMF restores the imbalance in the antioxidant response caused by HIV infection. HIV infection of human MDM reduces HO-1 and GPX1 expression, as assessed by Western blotting (A) and quantified by densitometry analysis (B). Values indicate mean ± SEM of six different donors. Statistical comparisons were made by two-tailed paired t test. C, DMF activates the Nrf2-dependent antioxidant response in HIV/MDM and restores HO-1 and GPX1 levels to that found in uninfected mock cells, as quantified by densitometry analysis (D). MMF activates the Nrf2-dependent antioxidant response in HIV/MDM and restores HO-1 and GPX1 levels to that found in uninfected mock cells (E), as quantified by densitometry analysis (F). Blots are representative of three independent experiments, with each replicate performed on cell preparations from different donors. Densitometry data are expressed as mean ± SEM and averaged from three different donors. *p < 0.05, **p < 0.01, ***p < 0.001 versus mock.

HO-1 does not mediate the attenuation of HIV replication or NF-κB signaling induced by DMF. A, SnMP, an inhibitor of HO-1 enzymatic activity, does not inhibit DMF-mediated attenuation of HIV replication. DMF and vehicle-treated HIV/MDM were exposed to 10 μM SnMP from days 6 to 15 postinfection. Culture supernatants were collected every 3 d and assessed for RT activity. RT curves are representative of three independent experiments, with each replicate performed on cell preparations from different donors. B, SnMP and CoPP, a specific inducer of HO-1, do not directly affect or alter DMF-mediated inhibition of the nuclear translocation of NF-κB proteins, as assessed by Western blotting. Human MDM were treated with 10 μM SnMP, 10 μM CoPP, and/or 100 μM DMF for 24 h before treatment with 1 ng/ml TNF-α (10 min) and subcellular fractionation. Western blot is representative of three independent experiments, with each replicate performed on cell preparations from different donors.

HO-1 induction reduces neurotoxin production in HIV/MDM without affecting HIV replication. SnMP, an inhibitor of HO-1 enzymatic activity, does not directly affect HIV replication (A) and supernatant from these SnMP-treated HIV/MDM is significantly more neurotoxic (B), despite equal levels of HIV replication. C, Uninfected mock/MDM and 20 nM EFZ-treated HIV/MDM, which normally produce minimal neurotoxins, are significantly more neurotoxic when treated with 10 μM SnMP. D, CoPP, a specific inducer of HO-1 expression, does not directly affect HIV replication and supernatant from these CoPP treated HIV/MDM is significantly less neurotoxic (E), despite high levels of virus replication. F, CoPP treatment exponentially increases HO-1 levels without greatly altering the other components of the antioxidant response, as assessed by Western blotting. For A and C, SnMP or CoPP was added at day 6 postinfection onwards and culture supernatants were collected every 3 d and assessed for RT activity. RT curves are representative of three independent experiments, with each replicate performed on cell preparations from different donors. For neuronal survival assays, survival was assessed by MAP2 ELISA and expressed as a percentage of untreated (UT) cultures (n = 6). Statistical comparisons were made by one-way ANOVA plus Newman–Keuls post hoc testing. Western blot is representative of three independent experiments, with each replicate performed on cell preparations from different donors. Two film exposures (short and long) of HO-1 are presented to demonstrate the extent of HO-1 induction over basal levels. ***p < 0.001 versus vehicle-treated paired condition.