Molecular Neurobiology

, Volume 55, Issue 2, pp 1352–1363 | Cite as

Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis

  • Biju Bhargavan
  • Georgette D. KanmogneEmail author


The recombinant HIV-1 CRF02_AG is prevalent in West-Central Africa but its effects on the blood-brain barrier (BBB) and HIV-associated neurocognitive disorders (HAND) are not known. We analyzed the effects of Tat from HIV-1 subtype-B (Tat.B) and CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major BBB component. Exposure of HBMEC to Tat.B increased IL-6 expression and transcription by 9- (P < 0.001) and 113-fold (P < 0.001), respectively, whereas Tat.AG increased IL-6 expression and transcription by 2.7–3.8-fold and 35.7-fold (P < 0.001), respectively. Tat.B induced IL-6 through the interleukin-1 receptor-associated kinase (IRAK)-1/4/mitogen-activated protein kinase kinase(MKK)/C-jun N-terminal kinase(JNK) pathways, in an activator protein-1(AP1)- and nuclear factor-kappaB (NFκB)-independent manner, whereas Tat.AG effects occurred via MKK/JNK/AP1/NFκB pathways. Tat-induced effects were associated with activation of c-jun (serine-63) and SAPK/JNK (Thr183/Tyr185). We demonstrated increased expression of transcription factors associated with these pathways (Jun, RELB, CEBPA), with higher levels in Tat.B-treated cells compared to Tat.AG. Functional studies showed that Tat.B and Tat.AG decreased the expression of tight junction proteins claudin-5 and ZO-1 and decreased the trans-endothelial electric resistance (TEER); Tat.B induced greater reduction in TEER, claudin-5, and ZO-1, compared to Tat.AG. Overall, our data showed increased inflammation and BBB dysfunction with Tat.B, compared to Tat.AG. This suggests these two HIV-1 subtypes differentially affect the BBB and central nervous system; our data provides novel insights into the molecular basis of these differential Tat-mediated effects.


HIV-1 Tat subtypes CRF02_AG Blood-brain barrier IL-6 JNK/NFκB signaling 



This work was supported by grant from the National Institute of Health, National Institute of Mental Health R01 MH094160.

Authors’ Contributions

B.B. carried out immunoassays, real-time PCR, Western blot, TEER, adhesion and migration assays and participated in the making of the figures and tables, data analysis, and writing the methods and results. G.D.K. conceived and designed the study, participated in the making of the figures and tables, data analysis, and wrote the manuscript.

Compliance with Ethical Standards

Conflicting Interests

The authors declare that they have no conflict of interest.


  1. 1.
    Antinori A, Arendt G, Becker JT, Brew BJ, Byrd DA, Cherner M, Clifford DB, Cinque P et al (2007) Updated research nosology for HIV-associated neurocognitive disorders. Neurology 69(18):1789–1799. doi: 10.1212/01.WNL.0000287431.88658.8b CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Banks WA, Ercal N, Price TO (2006) The blood-brain barrier in neuroAIDS. Curr HIV Res 4(3):259–266CrossRefPubMedGoogle Scholar
  3. 3.
    Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD (2006) Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J NeuroImmune Pharmacol 1(3):223–236. doi: 10.1007/s11481-006-9025-3 CrossRefPubMedGoogle Scholar
  4. 4.
    Bannwarth S, Gatignol A (2005) HIV-1 TAR RNA: the target of molecular interactions between the virus and its host. Curr HIV Res 3(1):61–71CrossRefPubMedGoogle Scholar
  5. 5.
    Gupta S, Mitra D (2007) Human immunodeficiency virus-1 tat protein: immunological facets of a transcriptional activator. Indian J Biochem Biophys 44(5):269–275PubMedGoogle Scholar
  6. 6.
    Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, Walczak H, Debatin KM, Krammer PH (1995) Sensitization of T cells to CD95-mediated apoptosis by HIV-1 tat and gp120. Nature 375(6531):497–500. doi: 10.1038/375497a0 CrossRefPubMedGoogle Scholar
  7. 7.
    Banks WA, Robinson SM, Nath A (2005) Permeability of the blood-brain barrier to HIV-1 tat. Exp Neurol 193(1):218–227. doi: 10.1016/j.expneurol.2004.11.019 CrossRefPubMedGoogle Scholar
  8. 8.
    Hudson L, Liu J, Nath A, Jones M, Raghavan R, Narayan O, Male D, Everall I (2000) Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues. J Neurovirol 6(2):145–155CrossRefPubMedGoogle Scholar
  9. 9.
  10. 10.
  11. 11.
    Anastassopoulou CG, Kostrikis LG (2006) Global genetic variation of HIV-1 infection. Curr HIV Res 4(3):365–373CrossRefPubMedGoogle Scholar
  12. 12.
    Robertson DLAJ, Bradac JA, Carr JK, Foley B et al (2000) HIV-1 nomenclature proposal. Science 288:55–56CrossRefPubMedGoogle Scholar
  13. 13.
    LANL (2016) HIV circulating recombinant forms (CRFs). HIV sequence database
  14. 14.
    Montavon C, Toure-Kane C, Liegeois F, Mpoudi E, Bourgeois A, Vergne L, Perret JL, Boumah A et al (2000) Most env and gag subtype A HIV-1 viruses circulating in west and west Central Africa are similar to the prototype AG recombinant virus IBNG. J Acquir Immune Defic Syndr 23(5):363–374CrossRefPubMedGoogle Scholar
  15. 15.
    Brennan CA, Bodelle P, Coffey R, Devare SG, Golden A, Hackett J Jr, Harris B, Holzmayer V et al (2008) The prevalence of diverse HIV-1 strains was stable in Cameroonian blood donors from 1996 to 2004. J Acquir Immune Defic Syndr 49(4):432–439. doi: 10.1097/QAI.0b013e31818a6561 CrossRefPubMedGoogle Scholar
  16. 16.
    PRB (2010) 2010 world population data sheet. The Population Reference Bureau, Washington DC
  17. 17.
    Woollard SM, Bhargavan B, Yu F, Kanmogne GD (2014) Differential effects of Tat proteins derived from HIV-1 subtypes B and recombinant CRF02_AG on human brain microvascular endothelial cells: implications for blood-brain barrier dysfunction. J Cereb Blood Flow Metab 34(6):1047–1059. doi: 10.1038/jcbfm.2014.54 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Chaudhuri A, Yang B, Gendelman HE, Persidsky Y, Kanmogne GD (2008) STAT1 signaling modulates HIV-1-induced inflammatory responses and leukocyte transmigration across the blood-brain barrier. Blood 111(4):2062–2072. doi: 10.1182/blood-2007-05-091207 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Yang B, Akhter S, Chaudhuri A, Kanmogne GD (2009) HIV-1 gp120 induces cytokine expression, leukocyte adhesion, and transmigration across the blood-brain barrier: modulatory effects of STAT1 signaling. Microvasc Res 77(2):212–219. doi: 10.1016/j.mvr.2008.11.003 CrossRefPubMedGoogle Scholar
  20. 20.
    Kanmogne GD, Schall K, Leibhart J, Knipe B, Gendelman HE, Persidsky Y (2007) HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across blood-brain barrier: implication for viral neuropathogenesis. J Cereb Blood Flow Metab 27(1):123–134. doi: 10.1038/sj.jcbfm.9600330 CrossRefPubMedGoogle Scholar
  21. 21.
    Maggirwar SB, Tong N, Ramirez S, Gelbard HA, Dewhurst S (1999) HIV-1 Tat-mediated activation of glycogen synthase kinase-3beta contributes to Tat-mediated neurotoxicity. J Neurochem 73(2):578–586CrossRefPubMedGoogle Scholar
  22. 22.
    Silvers JM, Aksenova MV, Aksenov MY, Mactutus CF, Booze RM (2007) Neurotoxicity of HIV-1 Tat protein: involvement of D1 dopamine receptor. Neurotoxicology 28(6):1184–1190. doi: 10.1016/j.neuro.2007.07.005 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Eugenin EA, King JE, Nath A, Calderon TM, Zukin RS, Bennett MV, Berman JW (2007) HIV-tat induces formation of an LRP-PSD-95- NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes. Proc Natl Acad Sci U S A 104(9):3438–3443. doi: 10.1073/pnas.0611699104 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zhou BY, Liu Y, Kim B, Xiao Y, He JJ (2004) Astrocyte activation and dysfunction and neuron death by HIV-1 Tat expression in astrocytes. Mol Cell Neurosci 27(3):296–305. doi: 10.1016/j.mcn.2004.07.003 CrossRefPubMedGoogle Scholar
  25. 25.
    Toborek M, Lee YW, Pu H, Malecki A, Flora G, Garrido R, Hennig B, Bauer HC et al (2003) HIV-Tat protein induces oxidative and inflammatory pathways in brain endothelium. J Neurochem 84. doi: 10.1046/j.1471-4159.2003.01543.x
  26. 26.
    Ranga U, Shankarappa R, Siddappa NB, Ramakrishna L, Nagendran R, Mahalingam M, Mahadevan A, Jayasuryan N et al (2004) Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine. J Virol 78(5):2586–2590CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Campbell GR, Watkins JD, Singh KK, Loret EP, Spector SA (2007) Human immunodeficiency virus type 1 subtype C Tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes. J Virol 81(11):5919–5928. doi: 10.1128/JVI.01938-06 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Carr JK, Salminen MO, Albert J, Sanders-Buell E, Gotte D, Birx DL, McCutchan FE (1998) Full genome sequences of human immunodeficiency virus type 1 subtypes G and A/G intersubtype recombinants. Virology 247(1):22–31. doi: 10.1006/viro.1998.9211 CrossRefPubMedGoogle Scholar
  29. 29.
    Tanaka T, Narazaki M, Kishimoto T (2014) IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol 6(10). doi: 10.1101/cshperspect.a016295
  30. 30.
    Tanaka T, Narazaki M, Ogata A, Kishimoto T (2014) A new era for the treatment of inflammatory autoimmune diseases by interleukin-6 blockade strategy. Semin Immunol 26(1):88–96. doi: 10.1016/j.smim.2014.01.009 CrossRefPubMedGoogle Scholar
  31. 31.
    Rossi J-F, Lu Z-Y, Jourdan M, Klein B (2015) Interleukin-6 as a therapeutic target. Clin Cancer Res 21(6):1248–1257. doi: 10.1158/1078-0432.ccr-14-2291 CrossRefPubMedGoogle Scholar
  32. 32.
    Bastard JP, Soulie C, Fellahi S, Haim-Boukobza S, Simon A, Katlama C, Calvez V, Marcelin AG et al (2012) Circulating interleukin-6 levels correlate with residual HIV viraemia and markers of immune dysfunction in treatment-controlled HIV-infected patients. Antivir Ther 17(5):915–919. doi: 10.3851/IMP2093 CrossRefPubMedGoogle Scholar
  33. 33.
    Hamlyn E, Fidler S, Stohr W, Cooper DA, Tambussi G, Schechter M, Miro JM, McClure M et al (2014) Interleukin-6 and D-dimer levels at seroconversion as predictors of HIV-1 disease progression. AIDS 28(6):869–874. doi: 10.1097/QAD.0000000000000155 CrossRefPubMedGoogle Scholar
  34. 34.
    Olwenyi OA, Naluyima P, Cham F, Quinn TC, Serwadda D, Sewankambo NK, Gray RH, Sandberg JK et al (2016) Brief report: differential associations of interleukin 6 and intestinal fatty acid-binding protein with progressive untreated HIV-1 infection in Rakai, Uganda. J Acquir Immune Defic Syndr 72(1):15–20. doi: 10.1097/QAI.0000000000000915 CrossRefPubMedGoogle Scholar
  35. 35.
    Kalayjian RC, Machekano RN, Rizk N, Robbins GK, Gandhi RT, Rodriguez BA, Pollard RB, Lederman MM et al (2010) Pretreatment levels of soluble cellular receptors and interleukin-6 are associated with HIV disease progression in subjects treated with highly active antiretroviral therapy. J Infect Dis 201(12):1796–1805. doi: 10.1086/652750 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Boulware DR, Hullsiek KH, Puronen CE, Rupert A, Baker JV, French MA, Bohjanen PR, Novak RM et al (2011) Higher levels of CRP, D-dimer, IL-6, and hyaluronic acid before initiation of antiretroviral therapy (ART) are associated with increased risk of AIDS or death. The Journal of infectious diseases 203(11):1637–1646. doi: 10.1093/infdis/jir134 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    French MA, Cozzi-Lepri A, Arduino RC, Johnson M, Achhra AC, Landay A (2015) Plasma levels of cytokines and chemokines and the risk of mortality in HIV-infected individuals: a case-control analysis nested in a large clinical trial. AIDS 29(7):847–851. doi: 10.1097/QAD.0000000000000618 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Borges ÁH, O’Connor JL, Phillips AN, Neaton JD, Grund B, Neuhaus J, Vjecha MJ, Calmy A et al (2016) Interleukin 6 is a stronger predictor of clinical events than high-sensitivity C-reactive protein or D-dimer during HIV infection. J Infect Dis 214(3):408–416. doi: 10.1093/infdis/jiw173 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kuller LH, Tracy R, Belloso W, De Wit S, Drummond F, Lane HC, Ledergerber B, Group ISS et al (2008) Inflammatory and coagulation biomarkers and mortality in patients with HIV infection. PLoS Med 5(10):e203. doi: 10.1371/journal.pmed.0050203 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Nordell AD, McKenna M, Borges ÁH, Duprez D, Neuhaus J, Neaton JD, the Insight Smart ESG, Committee SS (2014) Severity of cardiovascular disease outcomes among patients with HIV is related to markers of inflammation and coagulation. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease 3(3):e000844. doi: 10.1161/jaha.114.000844 CrossRefGoogle Scholar
  41. 41.
    Hsu DC, Ma YF, Hur S, Li D, Rupert A, Scherzer R, Kalapus SC, Deeks S et al (2016) Plasma IL-6 levels are independently associated with atherosclerosis and mortality in HIV-infected individuals on suppressive antiretroviral therapy. AIDS 30(13):2065–2074. doi: 10.1097/QAD.0000000000001149 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    McDonald B, Moyo S, Gabaitiri L, Gaseitsiwe S, Bussmann H, Koethe JR, Musonda R, Makhema J et al (2013) Persistently elevated serum interleukin-6 predicts mortality among adults receiving combination antiretroviral therapy in Botswana: results from a clinical trial. AIDS Res Hum Retrovir 29(7):993–999. doi: 10.1089/AID.2012.0309 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Gandhi N, Saiyed Z, Thangavel S, Rodriguez J, Rao KV, Nair MP (2009) Differential effects of HIV type 1 clade B and clade C Tat protein on expression of proinflammatory and antiinflammatory cytokines by primary monocytes. AIDS Res Hum Retrovir 25(7):691–699. doi: 10.1089/aid.2008.0299 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Kallunki T, Deng T, Hibi M, Karin M (1996) c-Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell 87(5):929–939CrossRefPubMedGoogle Scholar
  45. 45.
    Gupta S, Barrett T, Whitmarsh AJ, Cavanagh J, Sluss HK, Derijard B, Davis RJ (1996) Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J 15(11):2760–2770PubMedPubMedCentralGoogle Scholar
  46. 46.
    Akira S, Kishimoto T (1992) IL-6 and NF-IL6 in acute-phase response and viral infection. Immunol Rev 127:25–50CrossRefPubMedGoogle Scholar
  47. 47.
    Ambrosino C, Ruocco MR, Chen X, Mallardo M, Baudi F, Trematerra S, Quinto I, Venuta S et al (1997) HIV-1 Tat induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancer-binding protein beta (NF-IL6) transcription factors. J Biol Chem 272(23):14883–14892CrossRefPubMedGoogle Scholar
  48. 48.
    Ohno H, Kaneko S, Lin Y, Kobayashi K, Murakami S (1999) Human hepatitis B virus X protein augments the DNA binding of nuclear factor for IL-6 through its basic-leucine zipper domain. J Med Virol 58(1):11–18CrossRefPubMedGoogle Scholar
  49. 49.
    Nookala AR, Kumar A (2014) Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes. J Neuroinflammation 11(1):1–18. doi: 10.1186/s12974-014-0214-3 CrossRefGoogle Scholar
  50. 50.
    Shah A, Verma AS, Patel KH, Noel R, Rivera-Amill V, Silverstein PS, Chaudhary S, Bhat HK et al (2011) HIV-1 gp120 induces expression of IL-6 through a nuclear factor-kappa B-dependent mechanism: suppression by gp120 specific small interfering RNA. PLoS One 6. doi: 10.1371/journal.pone.0021261
  51. 51.
    Gangwani MR, Kumar A (2015) Multiple protein kinases via activation of transcription factors NF-κB, AP-1 and C/EBP-δ regulate the IL-6/IL-8 production by HIV-1 Vpr in astrocytes. PLoS One 10(8):e0135633. doi: 10.1371/journal.pone.0135633 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Toborek M, Lee YW, Flora G, Pu H, Andras IE, Wylegala E, Hennig B, Nath A (2005) Mechanisms of the blood-brain barrier disruption in HIV-1 infection. Cell Mol Neurobiol 25(1):181–199CrossRefPubMedGoogle Scholar
  53. 53.
    Dallasta LM, Pisarov LA, Esplen JE, Werley JV, Moses AV, Nelson JA, Achim CL (1999) Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis. Am J Pathol 155(6):1915–1927CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Boven LA, Middel J, Verhoef J, De Groot CJ, Nottet HS (2000) Monocyte infiltration is highly associated with loss of the tight junction protein zonula occludens in HIV-1-associated dementia. Neuropathol Appl Neurobiol 26(4):356–360CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Pharmacology and Experimental NeuroscienceUniversity of Nebraska Medical CenterOmahaUSA

Personalised recommendations