HIV-1 associated neuroinflammation : effects of two complimentary medicines illustrated in an in vitro model of the blood-brain barrier

Africa, Luan Dane (2014-12)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Background: Neuroinflammation is central to the aetiology of HIV-associated neurocognitive disorders (HAND) that are prevalent in late stage AIDS. ARV treatments are rolled out relatively late in the context of neuroinflammatory changes, so that their usefulness in directly preventing HAND is probably limited. It is common practice for HIV+ individuals in developing countries to make use of traditional/complimentary medicines. One such medicine is Sutherlandia frutescens - commonly consumed as a water infusion. We have also identified a new candidate complimentary medicine for use in this context - grape seed-derived proanthocyanidolic oligomers (PCO) have significant anti-inflammatory action in the peripheral compartment in the context of e.g. skeletal muscle injury, but have not been investigated in the context of either neuroinflammation or HIV/AIDS. Here the efficacy of these two substances as an anti-inflammatory modality in this context was investigated in an in vitro co-culture model of the blood-brain barrier (BBB). Methods: Single cultures of human astrocytes, HUVECs and primary human monocytes, as well as co-cultures (BBB), were stimulated with HIV-1 subtype B & C Tat protein and/or HL2/3 cell secretory proteins after pre-treatment with S. frutescens or PCO extracts. Effects of this pre-treatment on pro-inflammatory mediator expression and monocyte migration across the BBB were assessed. Results: In accordance with others, B Tat was more pro-inflammatory than C Tat, validating our model. S. frutescens decreased IL-1β secretion significantly (P<0.0001), but exacerbated both monocyte chemoattractant protein-1 (P<0001) – a major role player in HIV-associated neuroinflammation – and CD14+ monocyte infiltration across the BBB (P<0.01). PCO pre-treatment resulted in a significantly dampened IL-1β (P<0.0001) response to stimulation with HIV-associated proteins. In contrast to S. frutescens, PCO modulated monocyte chemoattractant protein-1 (P<0001) response and decreased capacity for CD14+ monocytes to migrate across the simulated BBB (P<0.0001). Additionally, PCO pre-treatment decreased both GFAP (P<0.001) and HSP-27 (P<0.001) expression in the astrocytes of the BBB. Conclusions: Current data illustrates that the combined use of HL2/3 cells and the simulated BBB presents an accurate, disease relevant in vitro model with which to study neuroinflammation in the context of HIV/AIDS. In addition, our results caution against the use of S. frutescens as anti-inflammatory modality at any stage post-HIV infection. Novel data presented here illustrate that PCO is able to blunt the MCP-1 and IL-1β response to HIV-1 proteins in single cultures of human astrocytes and HUVECs, as well as in an in vitro simulation of the BBB. In addition, PCO was able to limit monocyte transmigration across the simulated BBB in response to HIV-1 proteins generated by HL2/3 cells. This suggests that grape seed-derived PCO could be considered as complimentary anti-neuroinflammatory drug in the context of HIV/AIDS.

AFRIKAANSE OPSOMMING: Agtergrond: Neuroinflammasie staan sentraal in die ontwikkeling van MIV-verwante toestande wat gekenmerk word deur neurokognitiewe afteruitgang, veral in die later stadia van die siekte. Aangesien anti-virale middels relatief laat toegedien word in die konteks van neuroinflammasie, is hul rol in die voorkoming van neuroinflammatoriese veranderinge heel moontlik weglaatbaar. MIV+ individue, veral in ontwikkelende lande, gebruik algemeen natuurlike medisinale preparate. Sutherlandia frutescens is een so „n middel wat as „n tee ingeneem word. Verder het ons ook „n nuwe kandidaat komplimentêre medisyne identifiseer – druiwepitekstrak wat polifenole bevat (PCO) het aansienlike anti-inflammatoriese eienskappe in die periferie, bv. in die konteks van skeletspierskade, maar die middel is nog nie voorheen in die konteks van neuroinflammasie of MIV/VIGS ondersoek nie. Hier word die anti-inflammatoriese effektiwiteit van beide middels in hierdie konteks ondersoek deur gebruik te maak van „n in vitro simulasie van die bloedbreinskans (BBS). Metodes: Kulture van menslike astrosiete, menslike naelstring endoteelselle (HUVECs) en primêre menslike monosiete, sowel as gesamentlike kulture (BBS) is met MIV-1 subtipe B en C Tat proteïen en/of HL2/3 selprodukte gestimuleer na voorafbehandeling met S. frutescens of PCO ekstrakte. Effekte op pro-inflammatoriese mediator uitdrukking sowel as monosiet migrasie oor die BBS is ondersoek. Resultate: In ooreenstemming met die literatuur was B Tat meer inflammatories as C Tat, wat die akkuraatheid en gepastheid van ons model bevestig. . S. frutescens het afskeiding van IL-1β betekenisvol verminder (P<0.0001), maar het afskeiding van beide monosiet chemoaantrekkingsproteïen-1 – „n groot rolspeler in MIV-verwante neuroinflammasie – en CD14+ monosiet migrasie oor die BBS vererger (P<0.0001 en P<0.01 onderskeidelik). PCO behandeling het „n betekenisvolle demping van die IL-1β reaksie (P<0.0001) op stimulasie met MIV-geassosieerde proteïene tot gevolg gehad. Anders as S. frutescens het PCO die MCP-1 reaksie, asook CD14+ monosiet migrasie betekenisvol inhibeer. Verder het PCO ook beide GFAP en HSP-27 uitdrukking in astrosiete van die BBS verminder (beide P<0.001). Gevolgtrekkings: Huidige data wys dat die gekombineerde gebruik van HL2/3 selle en die gesimuleerde BBS „n akkurate en fisiologies relevante in vitro model daarstel, waarmee neuroinflammasie in die konteks van MIV/VIGS bestudeer kan word. Ons resultate waarsku verder teen die gebruik van S. frutescens as anti-inflammatoriese middel in selfs die vroeë stadium na MIV infeksie. Oorspronklike data wat hier aangebied word illustreer dat PCO die pro-inflammatoriese reaksie op MIV-proteïene in kulture van astrosiete en HUVECs, asook die in vitro simulasie van die BBS, effektief demp. Verder het PCO die vermoë getoon om monosiet migrasie oor die BBS, in reaksie op MIV-1 proteïene wat hul oorsprong uit HL2/3 selle het, te beperk. Hierdie bevindings beteken dat PCO dus eerder as S. frutescens oorweeg moet word as komplimentêre anti-inflammatoriese medisyne in die konteks van MIV/VIGS.

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