Anti-inflammatory cellular targets on neutrophils elucidated using a novel cell migration model and confocal microscopy: a clinical supplementation study

Arguably one of the most important and unique aspects of inflammation, is the ability of phagocytic immune cells to migrate through tissue to the site of injury or pathogenic presence. Efficient, directionally accurate movement of phagocytes to a site of injury will result in faster repair by the minimum number of tissue-infiltrating immune cells. This will not only lead to faster resolution of inflammation, but will also result in less secondary damage to otherwise healthy tissue (and thus milder clinical symptoms of inflammation), as approximately 80% of secondary tissue injury has been ascribed to the normally excessive response of neutrophils [1], which includes the release of reactive oxygen species and proteases [2]. Also, in terms of mononuclear phagocytes, macrophage phenotype M1 is recognised as a pro-inflammatory phenotype capable of migration into tissue (thus potentially exacerbating inflammation), while the more anti-inflammatory M2 phenotype seems unable to leave circulation to infiltrate tissue [3‐5].

Grape seed-derived products – and polyphenols (GSP) in particular – have received much attention in terms of its beneficial effects as anti-oxidant [6, 7], although it is also known to have anti-inflammatory effects [8, 9]. Since the anti-inflammatory effects of grape-derived products have received relatively less attention in terms of research, despite the well-known links between oxidative stress and inflammation, this was a particular focus of the current study.

Despite several recent reviews highlighting the many benefits to be derived from grape-derived products [9, 10], the majority of data reported was from pre-clinical models. In fact, in terms of human clinical results, data on the anti-inflammatory effects of GSP is extremely limited due to the recognised difficulty of translating natural product benefits from animal models to human models in research [10]. We have previously shown in pre-clinical investigations in rodents, that GSP altered neutrophil migration into injured tissue, thereby limiting secondary damage and the magnitude of the total inflammatory response [7, 11, 12]. A second effect associated with GSP treatment in vivo in rats, was the predominance of the more anti-inflammatory M2 phenotype [12]. Also, in an in vitro model of HIV-associated neuroinflammation, GSP was shown to limit both pro-inflammatory cytokine secretion and chemotaxin-induced human monocytic cell migration across the blood brain-barrier [13]. Together, these data suggest that GSP targets specific cellular sites to beneficially alter the course of inflammation. However, to our knowledge, these effects have not been investigated in an in vivo human model and information on specific molecular targets of GSP remain limited.

Therefore, the purpose of the current study was to expand on available knowledge by conducting an in vivo supplementation study in human subjects. We hypothesised that a 2-week supplementation with GSP would improve the directional accuracy of neutrophils in response to a chemotactic signal, via modulation of proteins involved in the movement of these phagocytes. In addition to our primary aim of investigating the effect of GSP supplementation on cellular role players facilitating neutrophil chemotactic movement, a second aim was the design and implementation of a novel cell migration assay with which to measure both functional outcome (cell migration) and mechanisms (molecular role players) in parallel and in the same cells, so that functional capacity may be correlated with altered expression or activation of cellular targets.

Although the data presented is statistically underpowered due to low subject number and high inter- and intra-individual variability, it does provide some insights which may inform on future study design. Current data firstly present a novel method by which in vitro cell migration may be assessed in parallel for both functional capacity and molecular cellular role players, using the exact same cell sample. Secondly, data suggest a role for GSP modulation of cellular role players involved in neutrophil chemotactic movement that is in line with results from animal models and thus warrants further investigation. No effect on migration was observed for GSP treatment. Although several reports of positive effects of polyphenols and GSP on inflammation exist [7, 11, 12] there is relative paucity of information on neutrophil migration and chemokinesis after in vivo GSP supplementation in humans. In contrast to reported in vitro results, current data showed high variability, which may be attributed to different factors, of which at least some are related to study design. Firstly, neutrophils which are isolated and then treated with GSP are exposed to a more controlled environment (in terms of dose of GSP, time of exposure and potential confounding factors), which allows for direct effects of GSP to be detected more readily. In contrast, in in vivo studies, the net effects of GSP is assessed after the supplement has been metabolised – a process which depends on the inherent metabolic rate of each individual assessed. Furthermore, the neutrophil as the first line of defence is arguably the most excitable immune cell, so that it may be activated during the supplementation period for various reasons unrelated to the study intervention. Thus, the relatively larger inter- and intra-individual variations may mask effects.

In order to better understand the variation, a reliability value calculation was performed on both total and linear distance, both between individuals and for the same individuals over time. Briefly, reliability was calculated – the inter-individual repeatability value was 0.61 and 0.43 for total and linear distance respectively, and the intra-individual repeatability value 0.39 and 0.57. This implies that inter-person variation contributed to 61% and 43% of the variability in current data for these two parameters. Statistical power could therefore be improved by addition of more subjects, or increasing the effect size (i.e. using a population more in need of anti-oxidant or anti-inflammatory modulation, such as an aged or inflamed population). A subsequent power analysis indicated that more than 80 subjects would be required to provide sufficient statistical power – a number that is not feasible, given the high cost of analyses. Furthermore, given the fact that day-to-day variation is shown to contribute 39 and 57% of variability for total and linear distance respectively, even data from a bigger group is likely to be complex to interpret in a longitudinal study design. Rather, we recommend that in vivo studies should either be of cross-sectional design – i.e. focus on acute effects on potential molecular targets – or that a very controlled clinical trial should be conducted, during which not only food intake, but also exposure to potential antigens, can be limited. Alternatively, a population with a poorer baseline inflammatory profile may be more ideal to illustrate anti-inflammatory effects, as the expected effect size would be larger. Therefore, although no significant effect was seen in terms of neutrophil movement parameters, given this variability, it does not exclude that GSP may have effects at molecular level.

Since GSP had no significant effect on neutrophil chemokinesis in the healthy population in this study – who probably had limited reserve for improvement of oxidative stress or inflammatory status – these results cannot conclusively inform on effects of GSP on neutrophil migration. Using conventional techniques, i.e. migration assessment in a closed system, the conclusion may have incorrectly been made that GSP had no effect on migration. However, the immunochemistry results obtained from the migrating cells suggest differently, significantly altering the interpretation here.

For example, the consistent aberration in ROCK-related parameters in the GSP-treated group, suggests modulatory effect of GSP on neutrophil cellular parameters in the context of the ROCK-PI3K-PTEN pathway. Neutrophil polarization is a process that causes neutrophils to undergo a mechanical shape change (elongation) which demonstrate a clear leading and tail end of the cell [15]. In terms of the role players assessed in this context, we hypothesised that GSP may have its effect by separating localisation sites of PI3K (to the front) and ROCK (to the rear) in a migrating cell, to improve overall migration capacity and directional accuracy. Futhermore, in terms of the literature on changes in ROCK expression, a decrease has been linked to increased recruitment and migration of neutrophils [16]. In contrast, the loss of ROCK has been associated with neutrophil tail retraction defects [17, 18], suggesting an optimal level of ROCK expression is required for maximum migration capacity. Current data suggests that GSP may modify the ROCK response to fMLP stimulation, although more research is required for a firm conclusion to be made. This is in line with our hypothesis, and may further suggest that GSP may act as such an “optimising modulator” of ROCK. ROCK is known to regulate PTEN – whose role is to dephosphorylate PIP3 to PIP2 and maintain the anterior-posterior PIP3 gradient [19], which is vital for maintaining directional accuracy [20, 21] and its expression level is positively associated with neutrophil migration and the recruitment of neutrophils [22, 23]. Furthermore, current results suggest that a greater extent of co-localization of ROCK with PI3K may occur under stimulated conditions than under unstimulated conditions (≈25%, ANOVA main effect, P < 0.05). Interestingly, the same sensitivity was not seen for PI3K. Although the isoform of PI3K assessed in the current study – p110β – has been implicated in cell migration [24], the p110γ isoform has been demonstrated to be more sensitive than the p110β to experimental G protein-coupled receptor mediated activation [25], which may account for the seemingly lower sensitivity of PI3K to ROCK here. This is a limitation of the current study and should be improved on in subsequent trials to ensure optimal sensitivity and relevance. No other literature is available on PI3K and ROCK expression on actively migrating neutrophils, but in light of the increased co-localisation of these parameters after fMLP stimulation, data may suggest increased capacity for cell polarisation after GSP treatment. Importantly, this supports our interpretation that GSP may not necessarily affect total ROCK or PI3K expression levels, but may rather optimise the sites in the cell where they are localised. The decrease in ROCK localisation with PI3K may indeed point towards an improved, GSP-associated polarisation of the cell to have ROCK focused in the rear and PI3K focused in the leading edge of the cell, which should improve migratory economy, as movement will be more directed.

In terms of cell adhesion, plasma levels of soluble adhesion molecules is the more commonly used protocol for assessing cellular adhesion dynamics in the recent literature. An obvious limitation of this approach is that the source of adhesion molecules in plasma cannot be deduced from this, since the shedding of expression markers can be attributed to various cell types. Results in the present study indicated that GSP does not have a significant effect on cellular expression of CD66b, ICAM-1 or VCAM-1. Although one would expect an anti-inflammatory product to exhibit significant modulatory effects on adhesion, our result is in line with the data of Schleimer et al. [26], who similarly reported no change in neutrophil adhesion molecule expression after treatment with IL-4, which is also a known anti-inflammatory modality [27]. In contrast, an in vitro study on the effects of red grape polyphenols on some of these markers [28], reported decreased ICAM-1 protein and gene expression. However, this effect of the polyphenols were only evident after stimulation of the (HUVEC) cells to lipopolysaccharide (LPS), which is a known up-regulator of these adhesion molecules [29‐31]. Together, these results suggest that assessment of anti-inflammatory effects under basal conditions is not reflected by cellular adhesion protein expression, and thus that plasma adhesion molecule expression modulation may then reflect modulation of adhesion molecule expression at the level of the endothelium. This in itself is interesting, for two reasons. Firstly, increased lymphocyte adhesion molecule expression has been linked to hypercoagulability and increased risk for atherosclerosis [32]. Secondly, GSP has been reported to decrease VCAM-1 expression [8, 33]. Together, this suggests that while cell adhesion are not compromised – i.e. immune function is maintained in this context – GSP may have additional benefit in the context of atherosclerosis by modification of the endothelium itself. This opens up a new avenue for investigation in the preventative medicine niche.

Finally, our result indicate for the first time in humans, that GSP treatment may indeed exert direct effects on peripheral monocytic cells to limit inflammatory infiltration into tissue, a result we have previously reported in rats with acute skeletal muscle damage-associated inflammation [7, 11, 12] as well as in an in vitro simulation of human HIV-associated inflammatory infiltration across the blood brain barrier [13]. Specifically, the significant inverse correlation between PRE and POST expression levels of both CD274 and MPO within the GSP group only, suggests an adaptogenic role for GSP – i.e. effectively being able to decrease relatively higher M1 protein expression levels while increasing relatively lower levels. These results, reported under unstimulated conditions, are especially remarkable in terms of the potential effect of GSP, if one considers that significant changes in M1/M2 markers have only ever been reported after stimulation of cells [34]. Interestingly, type M1 macrophages are known secrete pro-inflammatory cytokines as well as ROS. In excess, ROS can be extremely detrimental to the immune cell itself [35]. Hence, this suggested modulation of M1 phenotype expression by GSP is of importance, as it suggest a potential additional avenue by which GSP may indirectly exert its anti-oxidant effect.

In conclusion, we provide the first evidence of an immunomodulatory role for GSP in a human in vivo supplementation model, in support of data from animal models. The limited beneficial effects already evident in this pilot study in a healthy, young population – and the absence of any adverse effects – warrants further investigation of GSP via an appropriately powered clinical trial in more compromised populations, such as in aged or chronically inflamed individuals.