Elucidation of molecular mechanisms that may contribute to polyphenol-induced effects on neutrophil chemokinesis

Smith, Tanya (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Grape-derived products are high in polyphenols and are known to have anti-oxidant and anti-inflammatory effects. In vivo studies in the context of muscle injury-induced inflammation have proven grape seed-derived proanthocyanidin oligomers (PCO) to benefit recovery by modulation of neutrophil infiltration into damaged tissue, thereby reducing secondary damage. Also, in these models, PCO have been shown to facilitate an early anti-inflammatory macrophage phenotype shift, which resulted in faster resolution of inflammation and shortened recovery time. However, these results have not been investigated in a human model and the specific molecular targets of PCO is not clear. This study therefore aimed to investigate potential molecular targets of PCO in a normally healthy population. In addition, given anecdotal concern about consumer safety, a secondary aim was to investigate haematological effects of short-term PCO supplementation in humans. A limited haematology assessment was included to address these concerns. Eighteen normally healthy volunteers between the ages of 18-25 years old (13 female and 5 male) were subjected to daily oral supplementation with 140mg of PCO for a 2-week period. Blood samples were taken at baseline (day 0), as well as on days 7 and 14. Day 0 and 14 samples were comprehensively analysed for in vitro neutrophil chemokinetic capacity towards a chemotaxin (fMLP) using live cell tracking software, as well as neutrophil expression levels of adhesion molecules (ICAM-1, VCAM-1 and CD66b) by flow cytometry and cell polarisation factors (ROCK, PI3K) using immunohistochemistry. Macrophage expression of markers indicative of different phenotypes were assessed using flow cytometry. In addition, day 7 samples were assessed for PCO-induced effects on general haematology and hemostasis. No adverse effects of PCO was evident. A novel neutrophil migration assay was developed to allow immunohistochemistry staining on chemokinetic neutrophils, allowing assessment of molecular role players involved in chemokinesis and actual movement capacity on the same cells. Although PCO supplementation had no evident effect on neutrophil chemokinesis or adhesion molecule expression, the increase in ROCK co-localisation with PI3K under stimulated conditions was prevented, while ROCK expression itself tended to be decreased. Macrophage phenotype markers CD274 and MPO – both indicative of a pro-inflammatory M1 phenotype – was normalised after PCO treatment. We conclude that the PCO product employed in the current study, was safe for consumption. Furthermore, data indicates that neutrophil chemokinetic capacity may be optimised by PCO via modulation of the ROCK-PI3K-PTEN system, which results in better front-rear synchronisation of cell polarisation and thus movement. Finally, data confirms earlier reports in rodents, of a direct effect on macrophages to achieve a relatively anti-inflammatory phenotype predominance.

AFRIKAANSE OPSOMMING: Produkte afgelei van druiwe bevat hoë konsentrasies polifenole en is bekend vir hul anti-oksidant- en anti-inflammatoriese effekte. In vivo-studies oor spierbeskadiging en verwante inflammasie, het bewys dat druif-verwante polifenole (PCO) herstel stimuleer deurdat dit infiltrasie van neutrofiele na die beseerde weefsel beperk, wat sekondêre skade verminder. Hierdie modelle het ook gewys dat PCO ‘n vroëë anti-inflamatoriese fenotipeverskuiwing in makrofage bewerkstelling, wat lei tot ‘n vinniger afname in inflammasie en ‘n verkorte hersteltyd. Hierdie effekte is egter nog nie voldoende in mensmodelle nagevors nie en die spesifieke molekulêre teikens is onbekend. Hierdie studie het gepoog om die teikens van PCO in ‘n gesonde populasie te ondersoek. Gegewe berigte van gebruikerskommer rakende veiligheid, was ‘n verdere doel om die effek van korttermyn supplementasie met PCO op die algemene hemotologie and hemostase te ondersoek. Agtien gesonde vrywilligers, tussen die ouderdomme van 18-25 jaar (13 vroulik en 5 manlik), is onderwerp aan ‘n daaglikse mondelinge aanvulling van 140mg PCO oor ‘n tydperk van twee weke. Bloedmonsters is geneem voor supplementasie (dag 0), asook na 7 en 14 dae van aanvullling. Dag 0 en 14 monsters is deeglik ondersoek vir in vitro neutrofiel chemokinetiese bewegingskapasiteit in die rigting van ‘n chemotaksin (fMLP) deur van lewendesel-mikroskopie asook neutrofiel uitdrukkingsvlakke van adhesiemolekule (ICAM-1, VCAM-1 en CD66b), deur vloeisitometrie en selpolarisasiefaktore (ROCK, PI3K) met immunohistochemie gebruik te maak. Makrofaaguitdrukking van merkers wat verskilledne fenotipes aandui is met vloeisitometrie bepaal. Dag 7 monsters is ontleed vir PCO-verwante effekte op algemene hemotologie en hemostatse-aanwysers. Geen newe-effekte van PCO inname was waarneembaar nie. ‘n Nuwe neutrofielmigrasietoets is ontwikkel om immunohistochemiese ondersoeke op migrerende neutrofiele te kon uitvoer, om die bewegingskapasiteit en molekulêre rolspelers in dieselfde selle te assesseer. Alhoewel PCO aanvullings geen sigbare effek op neutrofielbeweging of die uitdrukking van adhesiemolekule gehad het nie, was ROCK-uitdrukking op neutrofiele laer, terwyl die verhoodge gesamentlike uitdrukking van ROCK met PI3K, wat gepaard gaan met stimulasie, betekenisvol ge-inhibeer is. Die makrofaag merkers CD274 en MPO, beide aanwysers van die pro-inflammatoriese M1 fenotipe, het na PCO aanvulling genormaliseer. Die gevolgtrekking is dat die PCO-produk veilig is vir gebruikers. Verder dui data aan dat neutrofiel-chemotaktiese kapasiteit ge-optimaliseer word deur PCO, deurdat dit die ROCK-PI3K-PTEN sisteem moduleer, om sodoende beter voorpunt-agterpuntsinchronisasie van selpolarisasie en dus beweging, teweeg te bring. Die data verkry in hierdie studie onderskraag vroëëre eksperimentele dat verkry in knaagdiere, van ‘n direkte effect van PCO op makrofage sodat die anti-inflammatoriese fenotipe oorheers.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100953
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