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Using a simulated blood-brain barrier to investigate potential modulators of HIV-1-associated neuro-inflammatory processes in vitro
... 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. ...
... 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 Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH ("Springer Nature"). ...
Background
In vivo studies have shown grape seed-derived polyphenols (GSP) to benefit in recovery from muscle injury by modulation of neutrophil infiltration into damaged tissue, thereby reducing secondary damage, as well as by facilitating an early anti-inflammatory macrophage phenotype shift. The current study aimed to provide data in this context from human models and to elucidate specific molecular targets of GSP.Using a placebo-controlled, double-blind study design, eighteen normally healthy volunteers between the ages of 18–35 years old (13 female and 5 male) were orally supplemented with 140 mg/day of GSP for 2 weeks.Blood samples (days 0 and 14) were comprehensively analysed for in vitro neutrophil chemokinetic capacity towards a chemotaxin (fMLP) using a novel neutrophil migration assay, in combination with live cell tracking, as well as immunostaining for neutrophil polarisation factors (ROCK, PI3K) at migration endpoint. Macrophage phenotype marker expression was assessed using flow cytometry. ResultsfMLP induced significant chemokinesis (P < 0.01), validating our model. GSP did not exert a significant effect on neutrophil chemokinesis in this non-compromised population, but tended to decrease overall ROCK expression in fMLP-stimulated neutrophils (P = 0.06). Macrophage phenotype markers CD274 and MPO – indicators of a pro-inflammatory M1 phenotype – seemed to be normalised relative to baseline expression levels after GSP treatment. Conclusions
Current data suggest that GSP may have a modulatory effect on the ROCK-PI3K-PTEN system, but results in this normal population is not conclusive and should be confirmed in a larger, more inflamed population. Potential modulation of macrophage phenotype by GSP should be investigated further.
... However, the role of NO in the context of antioxidant status is much more complex, so that this effect of grape polyphenols should probably receive more attention before it can be interpreted fully in terms of mechanism(s) involved. Furthermore, recently in a coculture simulation of the human blood-brain barrier, another grape polyphenol, proanthocyanidin, was associated with significant inhibition of monocyte infiltration and proinflammatory cytokine secretion in HIV-associated neuroinflammation [64]. Such inhibition of neuroinflammation is associated with a better prognosis in terms of HIV-related neurodegeneration and dementia, further confirming the neuroprotective potential of grapederived antioxidants. ...
... Such inhibition of neuroinflammation is associated with a better prognosis in terms of HIV-related neurodegeneration and dementia, further confirming the neuroprotective potential of grapederived antioxidants. Taken together, these studies suggest that the neuroprotective effects of grape polyphenols involve both antioxidant and anti-inflammatory mechanisms, with the latter including not only modulated cytokine signalling, but also modulation of both motility and functional capacity of leukocytes, as previously illustrated by our group [64,65]. ...
Ageing is a pro-inflammatory state, caused in part by compromised neutrophil mobility and directional inaccuracy.1 The interlinked nature of oxidative stress and inflammation – especially in the context of ageing/accelerated ageing – is well documented.2 We have previously demonstrated potent anti-inflammatory activity of grape seed-derived polyphenols in the context of skeletal muscle damage3 and neuroinflammation.4 The aim of this study was to assess the effects of this natural product on ageing-related inflammation. HPLC analysis confirmed the grape (Vitis vinifera) seed extract to consist of mainly catechin and epicatechin (m/z 291), as well as proanthocyanidin B (m/z 579), and to have high anti-oxidant capacity when compared to other natural antioxidants in vitro. For example, the grape seed extract was able to reduce DPPH radicals in a manner similar to ascorbic acid (IC50 of 2.5 µg/ml vs. 2.1 µg/ml for ascorbic acid). Also, superoxide radicals generated by xanthine oxidase were scavenged in a dose-dependent manner by the grape seed extract (IC50 of 1.0 µg/ml vs. 10.9 µg/ml [ellagic acid] and 0.04 µg/ml [gallic acid]). Using a Dunn chamber and live-cell microscopy, the capacity of neutrophils isolated from young (< 25yr) and old (> 65yr) volunteers, to migrate towards a chemotactic signal (fMLP) was assessed (Figure 1). Neutrophils from old individuals (B) showed significantly less movement and greater directional inaccuracy than those of young individuals (A). This was corrected (C) by one hour of in vitro treatment with a grape seed-derived extract. In addition, grape seed extract-treated neutrophils had significantly lower intracellular myeloperoxidase content and significantly higher cell surface adhesion molecule expression when compared to controls. We conclude that the polyphenol extract from grape seeds may have beneficial effects on ageing both in terms of antioxidant and anti-inflammatory potential, specifically by modulation of neutrophil chemokinesis capacity.
... However, the role of NO in the context of antioxidant status is much more complex, so that this effect of grape polyphenols should probably receive more attention before it can be interpreted fully in terms of mechanism(s) involved. Furthermore, recently in a coculture simulation of the human blood-brain barrier, another grape polyphenol, proanthocyanidin, was associated with significant inhibition of monocyte infiltration and proinflammatory cytokine secretion in HIV-associated neuroinflammation [64]. Such inhibition of neuroinflammation is associated with a better prognosis in terms of HIV-related neurodegeneration and dementia, further confirming the neuroprotective potential of grapederived antioxidants. ...
... Such inhibition of neuroinflammation is associated with a better prognosis in terms of HIV-related neurodegeneration and dementia, further confirming the neuroprotective potential of grapederived antioxidants. Taken together, these studies suggest that the neuroprotective effects of grape polyphenols involve both antioxidant and anti-inflammatory mechanisms, with the latter including not only modulated cytokine signalling, but also modulation of both motility and functional capacity of leukocytes, as previously illustrated by our group [64,65]. ...
Advanced age is associated with increased incidence of a variety of chronic disease states which share oxidative stress and inflammation as causative role players. Furthermore, data point to a role for both cumulative oxidative stress and low grade inflammation in the normal ageing process, independently of disease. Therefore, arguably the best route with which to address premature ageing, as well as age-associated diseases such as diabetes, cardiovascular disease, and dementia, is preventative medicine aimed at modulation of these two responses, which are intricately interlinked. In this review, we provide a detailed account of the literature on the communication of these systems in the context of ageing, but with inclusion of relevant data obtained in other models. In doing so, we attempted to more clearly elucidate or identify the most probable cellular or molecular targets for preventative intervention. In addition, given the absence of a clear pharmaceutical solution in this context, together with the ever-increasing consumer bias for natural medicine, we provide an overview of the literature on grape (
Vitis vinifera
) derived products, for which beneficial effects are consistently reported in the context of both oxidative stress and inflammation.
... Assuming 100% absorption and bioavailability in an average-sized person with total body fluid volume of 15 l, this calculates to a dose of 10 ug/ml. Previous research by our group (Africa and Smith 2015) confirmed the suitability of this dose for in vitro studies in a series of cell viability tests on a variety of cell types. For treatment preparation, briefly, a stock of 100 ug/ml GSP was prepared in distilled water. ...
Oxidative stress and inflammation are intricately interlinked as aetiological factors in the context of ageing and chronic disease-related accelerated ageing. Previous research by our group has highlighted the anti-oxidant and anti-inflammatory potential of grape-derived polyphenols in the context of acute inflammation and oxidative stress. The aim here was to add to this by assessing efficacy of the treatment (acutely) to address ageing-associated cumulative pro-oxidant and pro-inflammatory changes in an in vitro model. Blood from young and aged humans was analysed for baseline oxidative stress and inflammatory status. Isolated neutrophils were acutely exposed to the polyphenol treatment in vitro. The chemokinetic capacity of treated and control neutrophils in response to fMLP was subsequently determined in a Dunn chamber, using live cell imaging. Neutrophils were also analysed for the expression of selected molecular markers associated with functional capacity and oxidative stress. Results indicate that the aged population had significantly worse oxidative stress and inflammatory profiles (higher plasma conjugated dienes and MPO) than young controls. Neutrophils isolated from both young and aged individuals had improved chemokinetic accuracy and capacity after in vitro polyphenol treatment. Additionally, increased shedding of CD16 and expression of CD66b suggested sites via which the polyphenol achieved improved neutrophil motility. We conclude that grape seed-derived polyphenols facilitated improved neutrophil functionality by acting on the molecular targets elucidated here.
... EBM was obtained from Lonza (#CC-3121), with additional compounds bought as a supplementary unit (Lonza, #CC-4133). Transendothelial migration was induced after seeding phagosome maturation arrested macrophages into 24 well inserts at 3 × 10 5 cells per insert and exposing cells to a chemotactic signal of 100 ng/ml monocyte chemoattractant protein 1 (MCP-1, Sigma-Aldrich, # SRP3109) in the bottom well, for 2.5 hr-a time we have previously established in a variety of similarmigration models as sufficient to allow for adequate quantifiable migration to take place(Africa & Smith, 2015a, 2015bKruger, Myburgh, & Smith, 2014). ...
One of the limiting factors in tissue regeneration, particularly in the context of chronic disease such as myodystrophy, motor neuron disease, sarcopenia and cardiovascular disease, is limited availability of stem cells. We propose employing autologous macrophages to deliver stem cells, thereby facilitating tissue regeneration, by a novel and relatively non-invasive therapeutic intervention. Circulatory monocytic cells of M1 phenotype have capacity for transendothelial migration to infiltrate damaged tissue, making them ideal delivery vehicles. However, in order to deliver viable stem cells these macrophages must undergo phagosome maturation arrest. Our aim was to induce phagosome maturation arrest in pre-polarised M1 macrophages, whilst maintaining capacity for phagocytic engulfment (including phagosome formation) and transendothelial migration. Primary human M1 macrophages were treated with a Wortmannin-Concanamycin A-Chloroquine cocktail to induce arrest. Modified cells were allowed to ingest 4.5μm protein-coated fluorescent latex beads (simulated stem cells), before migratory capacity in response to MCP-1 was assessed over a 2-hour period in a Transwell co-culture system. Data indicate that phagosome acidification (as indicated by pHrodo®) was prevented in treated cells, effectively limiting digestion of ingested “cargo” (1.23±0.26% vs. 7.52±0.98% in controls; p<0.0001). Neither phagocytic engulfment capacity (68.67±3.51% vs. 61.19±4.68%), nor migratory capacity (70.14±12.6 vs. 72.86±16.0 migrated cells per well) was compromised. We conclude that macrophages were successfully modified into transendothelial delivery vehicles, without compromising required functionality. This delivery system can be exploited to develop a novel method for focussed stem cell and/or drug delivery.
... Glucocorticoids are generally known to downregulate inflammatory processes by various routes, one of which is negative feedback terminating production and release of proinflammatory cytokines, such as TNF-α and IL-6 from various tissue cells. It is of interest that in stress-related conditions of varying severity, as in post-traumatic stress disorder (PTSD) and the metabolic syndrome, glucocorticoid-insensitivity is paralleled by a pro-inflammatory state, which is now generally recognised and accepted as aetiological factor in many chronic disease states, which include cardiovascular disease, cancer, diabetes and neurodegeneration (Africa & Smith, 2015a, 2015bCrawford, 2014;Das, 2002). ...
... Extrapolating our data to a clinical application, CNS infiltration of both HIV-1 infected and uninfected monocytes is one of the main routes by which the virus enters and seeds the CNS as a viral reservoir to initiate neuroinflammatory processes. Thus, in order for any anti-inflammatory modality to be useful in this context, it would need to modulate this response – as we have previously demonstrated for grape seed-derived polyphe- nols [28] – which S. frutescens does not, and which it in fact seems to aggravate. Thus, in our opinion the use of S. frutescens should be avoided in the presence of HIV infection, as our data suggests that S. frutescens may increase monocyte infiltration into the CNS of HIV patients . ...
Background:
Neuroinflammation is central to the aetiology of HIV-associated neurocognitive disorders (HAND) that are prevalent in late stage AIDS. Anti-retroviral (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 medicines. One such medicine is Sutherlandia frutescens - commonly consumed as a water infusion. Here its efficacy 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 (HA), 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 extract. Effects of this pre-treatment on pro-inflammatory cytokine secretion 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).
Conclusions:
Current data illustrates that the combined use of HL2/3 cells and the simulated BBB presents an accurate, physiologically 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.
With the effectiveness of therapeutic agents ever decreasing and the increased incidence of multi-drug resistant pathogens, there is a clear need for administration of more potent, potentially more toxic, drugs. Alternatively, biopharmaceuticals may hold potential but require specialised protection from premature in vivo degradation. Thus, a paralleled need for specialised drug delivery systems has arisen. Although cell-mediated drug delivery is not a completely novel concept, the few applications described to date are not yet ready for in vivo application, for various reasons such as drug-induced carrier cell death, limited control over the site and timing of drug release and/or drug degradation by the host immune system.
Here, we present our hypothesis for a new drug delivery system, which aims to negate these limitations. We propose transport of nanoparticle-encapsulated drugs inside autologous macrophages polarised to M1 phenotype for high mobility and treated to induce transient phagosome maturation arrest. In addition, we propose a significant shift of existing paradigms in the study of host-microbe interactions, in order to study microbial host immune evasion and dissemination patterns for their therapeutic utilisation in the context of drug delivery. We describe a system in which microbial strategies may be adopted to facilitate absolute control over drug delivery, and without sacrificing the host carrier cells.
We provide a comprehensive summary of the lessons we can learn from microbes in the context of drug delivery and discuss their feasibility for in vivo therapeutic application. We then describe our proposed “synthetic microbe drug delivery system” in detail. In our opinion, this multidisciplinary approach may hold the solution to effective, controlled drug delivery.
The modern lifestyle is characterised by various factors that cause accelerating ageing by the upregulation of oxidative stress and inflammation—two processes that are inextricably linked in an endless circle of self-propagation. Inflammation in particular is commonly accepted as aetiological factor in many chronic disease states, such as obesity, diabetes and depression. In terms of disease prevention or treatment, interventions aimed at changing dietary and/or exercise habits have had limited success in practise, mostly due to poor long-term compliance. Furthermore, other primary stimuli responsible for eliciting an oxidative stress or inflammatory response—e.g. psychological stress and anxiety—cannot always be easily addressed. Thus, preventive medicine aimed at countering the oxidative stress and/or inflammatory responses has become of interest. Especially in developing countries, such as South Africa, the option of development of effective strategies from plants warrants further investigation. A brief overview of the most relevant and promising South African plants which have been identified in the context of inflammation, oxidative stress and chronic disease is provided here. In addition, and more specifically, our group and others have shown considerable beneficial effects across many models, after treatment with products derived from grapes. Of particular interest, specific cellular mechanisms have been identified as therapeutic targets of grape-derived polyphenols in the context of inflammation and oxidative stress. The depth of these studies afforded some additional insights, related to methodological considerations pertaining to animal vs. human models in natural product research, which may address the current tendency for generally poor translation of positive animal model results into human in vivo models. The importance of considering individual data vs. group averages in this context is highlighted.
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