Dimethyl fumarate, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and macrophage-mediated neurotoxicity: a novel candidate for HIV neuroprotection

Abstract

Despite antiretroviral therapy (ART), HIV infection promotes cognitive dysfunction and neurodegeneration through persistent inflammation and neurotoxin release from infected and/or activated macrophages/microglia. Furthermore, inflammation and immune activation within both the CNS and periphery correlate with disease progression and morbidity in ART-treated individuals. Accordingly, drugs targeting these pathological processes in the CNS and systemic compartments are needed for effective, adjunctive therapy. Using our in vitro model of HIV-mediated neurotoxicity, in which HIV-infected monocyte-derived macrophages release excitatory neurotoxins, we show that HIV infection dysregulates the macrophage antioxidant response and reduces levels of heme oxygenase-1 (HO-1). Furthermore, restoration of HO-1 expression in HIV-infected monocyte-derived macrophages reduces neurotoxin release without altering HIV replication. Given these novel observations, we have identified dimethyl fumarate (DMF), used to treat psoriasis and showing promising results in clinical trials for multiple sclerosis, as a potential neuroprotectant and HIV disease-modifying agent. DMF, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and neurotoxin release. Two distinct mechanisms are proposed: inhibition of NF-κB nuclear translocation and signaling, which could contribute to the suppression of HIV replication, and induction of HO-1, which is associated with decreased neurotoxin release. Finally, we found that DMF attenuates CCL2-induced monocyte chemotaxis, suggesting that DMF could decrease recruitment of activated monocytes to the CNS in response to inflammatory mediators. We propose that dysregulation of the antioxidant response during HIV infection drives macrophage-mediated neurotoxicity and that DMF could serve as an adjunctive neuroprotectant and HIV disease modifier in ART-treated individuals.

FIGURE 1

Dimethyl fumarate and monomethyl fumarate attenuate HIV replication in human MDM. Human MDM infected with 50ng HIV (p24 ELISA, equivalent to 1.82 ± 0.22 kcpm/μL by reverse transcriptase (RT) activity assay) were treated with DMF (A) or MMF (B) over the course of infection at the indicated concentrations (1-30μM) or with 20nM of the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFZ). Culture supernatants were collected every 2-3 days, as indicated, and HIV replication was quantified by RT activity. C, DMF and D, MMF cause no cytotoxicity in HIV/MDM as assessed by LDH assay of supernatants harvested at day 14 post infection. Maximum (Max) LDH release represents the soluble LDH release following cell lysis. RT curves are representative of 3-4 independent experiments, with each replicate performed on cell preparations from different donors. LDH assays represent data averaged from 3-5 individual donors. All statistical comparisons were made by one-way ANOVA plus Newman-Keuls post hoc testing, ***p<0.001 vs. EFZ.

FIGURE 2

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 (1-30μM) during the course of infection. Neuronal survival was assessed by MAP2 ELISA and expressed as a percentage of untreated (UT) cultures (n = 6; ***p<0.001 vs. Vehicle). C, Representative images of rat cerebrocortical cultures immunofluorescently stained for MAP2 (red) and Hoescht 33324 (blue) following 24 hours treatment with the indicated HIV/MDM supernatant. Scale bar represents 50μm. All statistical comparisons were made by one-way ANOVA plus Newman-Keuls post hoc testing.

FIGURE 3

DMF inhibits NF-κB nuclear translocation, DNA binding and TNFα production in human MDM. A, DMF and B, MMF 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 hours, exposed to TNFα (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 PARP, a nuclear marker and loading control. Blots are representative of 4-6 independent experiments, with each replicate performed on cell preparations from different donors. C, DMF inhibits nuclear NF-κB p50 binding to DNA in TNFα stimulated MDM, as assessed by EMSA. Results of densitometry analysis were normalized to vehicle. D, DMF inhibits the production of TNFα in MDM stimulated with PHA (10μg/mL). Values are expressed as percent TNFα production relative to Vehicle treated cells (227 ± 11.9 pg/mL TNFα in Vehicle). Data are expressed as mean ± SEM and represent data averaged from 4 different donors. E, TNFα production in HIV/MDM is inhibited by DMF treatment. HIV/MDM were treated with DMF (1-30μM) or 20nM efavirenz (EFZ) over the course of infection and culture supernatants from day 14-15 post infection were assayed for TNFα by ELISA. Values represent the mean ± SEM of data averaged from 5 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 vs. Vehicle).

FIGURE 4

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 6 different donors. Statistical comparisons were made by two-tailed paired t-test (*p<0.05, **p<0.01 and ***p<0.001 vs. Mock). 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). E, MMF 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 (F). Blots are representative of 3 independent experiments, with each replicate performed on cell preparations from different donors. Densitometry data are expressed as mean ± SEM and represent data averaged from 3 different donors.

FIGURE 5

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 10μM SnMP from day 6 through day 15 post infection. Culture supernatants were collected every 3 days and assessed for RT activity. RT curves are representative of 3 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 10μM SnMP, 10μM CoPP and/or 100μM DMF for 24 hours before treatment with 1 ng/mL TNFα (10min) and subcellular fractionation. Western blot is representative of 3 independent experiments, with each replicate performed on cell preparations from different donors.

FIGURE 6

HO-1 induction reduces neurotoxin production in HIV/MDM without affecting HIV replication. A, SnMP, an inhibitor of HO-1 enzymatic activity, does not directly affect HIV replication and supernatant from these SnMP treated HIV/MDM are significantly more neurotoxic (B), despite equal levels of HIV replication. C, Uninfected Mock/MDM and 20nM efaverinz (EFZ) treated HIV/MDM, which normally produce minimal neurotoxins, are significantly more neurotoxic when treated with 10μM SnMP. D, CoPP, a specific inducer of HO-1 expression, does not directly affect HIV replication and supernatant from these CoPP treated HIV/MDM are 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 post infection onwards and culture supernatants were collected every 3 days and assessed for RT activity. RT curves are representative of 3 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; ***p<0.001 vs. vehicle treated paired-condition). Statistical comparisons were made by one-way ANOVA plus Newman-Keuls post hoc testing. Western blot is representative of 3 independent experiments, with each replicate performed on cell preparations from different donors. Two film exposures (short and long) of HO-1 are presented in order to demonstrate the extent of HO-1 induction over basal levels.

FIGURE 7

DMF and MMF reduce CCL2 induced chemotaxis in human monocytes. A, DMF and B, MMF inhibit CCL2-induced chemotaxis in freshly isolated human monocytes in a dose-dependent manner. Values are expressed as percent migration of unstimulated cells (US; 0 ng/mL CCL2) (n = 10-22; **p<0.01 vs. Vehicle). C, DMF decreases CCR2 expression on CD11b⁺CD14⁺ PBMCs following 6 hours of treatment, as quantified (D). E, DMF does not cause significant cell death over 6 hours of treatment in freshly isolated human monocytes, as measured by DAPI positivity in CD11b⁺CD14⁺ gated PBMCs. For all experiments, values represent data averaged from 3 different donors. All statistical comparisons were made by one-way ANOVA plus Newman-Keuls post hoc testing. Results of post test for linear trend are also presented.