In vivo effect of anti-inflammatory compounds on HIV-1 gp120 -mediated brain inflammation Tamima Ashraf, PhD candidate Supervisor: Dr. Reina Bendayan University of Toronto, Leslie Dan Faculty of Pharmacy, Toronto, Ontario, Canada
HIV-associated neurocognitive disorders: an evolving epidemic More than 34.2 million people living worldwide with HIV-1 infection (UNAIDS, 2011) 40 to 50% patients with HIV-1 infection develop neurological disorders From Vivithanaporn et al., 2010. Neurology.
HIV-1 induced brain inflammation Ashraf et al., 2012. Current pharmaceutical design.
Mrp4 P-gp P-gp MRP1 Mrp2 MRP4 MRP5 BCRP Mrp1 Mrp4 P-gp MRP1 Mrp5 MRP4 MRP5 Mrp6 MRP8 P-gp Bcrp Mrp3 MRP1 Mrp3 MRP4 MRP5 Mrp6 P-gp Bcrp Poor brain penetration of antiretrovirals: role of drug efflux transporters Astrocyte Neurovascular Unit Mrp6 Microglia Neuron Blood Endothelium P-glycoprotein (P-gp) Multidrug resistance associated proteins (MRP) Breast cancer resistance associated protein (BCRP) Brain Adapted from Ashraf et al., 2012.
Regulation of drug efflux transporters and cytokine secretion by gp120 in vitro gp120 induces secretion of TNF-α, IL-6 and IL-1β Interaction of gp120 and chemokine receptor CCR5 mediates cytokine secretion HIV-1 or gp120 gp120 gp120 TNF- or α or TNF-α IL6 CCR5 Neutralizing Antibody gp120 and cytokines affect expression of transporters HIV-1 or gp120 or IL6 decreases P-gp protein expression TNF- α increases P-gp protein expression gp120 or TNF- α increases Mrp1 protein expression TNF- α, IL-6, Mrp1 P-gp IL-1 β (Ashraf et al., 2011. J Neurosci Res.; Ronaldson et al., 2010. Mol Pharmacol.; Ronaldson and Bendayan,.2006. Mol. Pharmacol.)
Signaling pathways Mitogen activated protein kinase pathway (MAPK) Three main MAPK subfamilies: ERK1/2 JNKs P38Ks Nuclear factor kappab (NF-kB) Transcription factor Activated by pro-inflammatory cytokines, oxidative stress, viral or bacterial products Receptor activation Phosphorylation P65 P65 From Ronaldson PT, Persidsky Y, Bendayan R. 2008. Glia.
Potential anti-inflammatory compounds HIV-1/viral proteins/lps/ Amyloid-β MAPK, NF-κB Chloroquine Simvastatin Minocycline Curcumin Flurbiprofen Anti-malarial HMG-CoA reductase inhibitor Tetracyclinederivative Gene transcription of Pro-inflammatory cytokines Nucleus Cytosol Secretion of pro-inflammatory cytokines (Ho et al., 2011.AIDS Res Ther.; Sacktor et al., 2011.Neurology; Copeland and Brooks, 2010.J Infect Dis.)
Hypotheses During HIV-1 induced brain inflammation, signal transduction pathways (i.e., NF-κB, MAPK) are involved in i) secretion of pro-inflammatory cytokines and generation of oxidative stress ii) regulation of membrane drug efflux transporters in glial cells. Anti-inflammatory agents and inhibitors of signaling pathways can reverse HIV-associated cytokine secretion as well as oxidative stress in the brain.
Objectives 1. To implement an in vivo model of HIV-associated brain inflammation by intracerebroventricular administration of gp120 in rodents 2. To evaluate the potential effect of anti-inflammatory agents (i.e., chloroquine) in reversing the cytokine secretion and oxidative stress as well the disruption of BBB in our implemented rodent model of HIVassociated brain inflammation
Animal model Brain tissue and CSF Implantation of guide cannula Recovery ICV administration of gp120 (500ng) for 7 days ± Intraperitoneal administration of antiinflammatory compounds
Research Plan Gp120ADA (R5 tropic) injected rodent model +/- Intraperitoneal administration: Minocycline(50 to 25mg/kg) Chloroquine (25mg/kg) Simvastatin (1mg/kg) CSF collection Brain tissue collection (frontal cortex, striatum and hippocampus) Cytokine (TNF-α, IL-6 and IL1β ) ELISA analysis mrna Expression of TNF-α, IL1β, IL- 6 and Real time qpcr Protein expression of P-gp and MRP1 Immunoblotting Activation of glial cells (GFAP, Iba-1) Immunohistochemical analysis BBB Integrity Capillary isolation Immunoblotting
IL-1β mrna levels in the presence of anti-inflammatory compounds Frontal cortex Hippocampus Striatum * * * * ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ *p<0.05, compared to saline; ⱡ p<0.05, compared to gp120; n 10
mrna levels in the presence of anti-inflammatory compounds Frontal cortex Hippocampus Striatum * * * ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ ⱡ *p<0.05, compared to saline; ⱡ p<0.05, compared to gp120; n 10
TNF-α mrna levels in the presence of anti-inflammatory compounds Frontal cortex * * ⱡ *p<0.05, compared to saline; ⱡ p<0.05, compared to gp120; n 10
IL-1β and TNF-α secretion in the presence of anti-inflammatory compounds * CSF CSF * * ⱡ ⱡ ⱡ ⱡ *p<0.05, compared to saline; ⱡ p<0.05, compared to gp120; n 10
Effect of CCR5 inhibitor, Maraviroc, on IL-1β mrna levels Frontal cortex Hippocampus Striatum *p<0.05, compared to saline (n=12 for saline and gp120 groups; n=3 for HI gp120 and maraviroc treated groups)
Effect of Maraviroc on gp120-mediated secretion of IL-1β and TNF-α *p<0.05, compared to saline; n=6 *p<0.05, compared to saline; n=3
P-gp & Mrp1 protein expression in hippocampus P-gp Saline gp120_300ng MDA- MDR1 Mrp1 Saline gp120_500ng Hela- MRP1 Actin Actin * *p<0.05; n=6 for the groups compared above
Signaling pathways Mitogen activated protein kinase pathway (MAPK) Three main MAPK subfamilies: ERK1/2 JNKs P38Ks From Ronaldson PT, Persidsky Y, Bendayan R. 2008. Glia.
Saline gp120-mediated activation of MAPKs in vivo gp120_500ng ERK1/2 P-ERK1/2 Saline gp120_500ng JNKs P-JNKs Saline gp120_500ng P38K P-P38K *p<0.05; n=6 for the groups compared above No significant change in phosphorylation was observed in frontal cortex or striatum
Summary: characterization of in vivo model mrna expression P-gp protein expression Mrp1 protein expression Bcrp protein expression Frontal cortex IL-1β, and TNF-α No significant change Hippocampus IL-1β, No significant change Striatum IL-1β, No significant change To be completed CSF IL-1β, TNF-α
Summary: effect of anti-inflammatory compounds Chloroquine +gp120 Minocycline +gp120 Simvastatin +gp120 Frontal cortex IL-1β, IL-1β,, TNF-α Hippocampus IL-1β, IL-1β,, TNF-α IL-1β, Striatum IL-1β, IL-1β, IL-1β, CSF IL-1β IL-1β, TNF-α _
Future work To implement an in vivo model of HIV-1 gp120- mediated cognitive deficits To investigate the anti-inflammatory compounds in preventing gp120 induced cognitive deficits
Significance High prevalence of neurological disorders and lower survival rate among patients with HIV-1 infection Understanding mechanisms behind ARV drug resistance and brain inflammation may provide novel therapeutic approaches to benefit patients with HIVassociated neurological complications Identifying well-tolerable anti-inflammatory compounds that demonstrate better brain permeability has the potential to be used as adjuvant therapy in treatment/prevention of neurocognitive disorders
Acknowledgements Supervisor: Dr. Reina Bendayan Advisory committee members: Dr. Stephane Angers Dr. Peter Pennefather Dr. Lyanne Schlichter Collaborators: Dr. Yuri Persidsky, Temple University, Philadelphia Dr. Michelle Farrugia, Mount Sinai Hospital, Toronto Cryostat/microscopy training: Dr. Jeffrey Henderson, University of Toronto Past lab member: Dr. Patrick Ronaldson Leslie Dan Faculty of Pharmacy, University of Toronto Present lab members: Dr. Wenlei Jiang Dr. Min Rui Blake Ziegler Other members of Bendayan s lab
Summary gp120 Chloroquine +gp120 Minocycline +gp120 Simvastatin +gp120 Frontal cortex IL-1β,, TNF-α IL-1β, IL-1β,, TNF-α Hippocampus IL-1β, IL-1β, IL-1β, IL-1β, Striatum IL-1β, IL-1β, IL-1β, IL-1β, CSF IL-1β IL-1β IL-1β Not determined TNF-α TNF-α TNF-α TNF-α