The Role of Wine in Modulating Inflammatory Processes: A Review

Several epidemiological studies associated the consumption of wine with the reduction of the risk factors for cardiovascular disease and certain cancers, as well as for diabetes. These conditions are characterized by inflammatory mechanisms in addition to other biological mechanisms. Acute and chronic inflammation is mediated by a plethora of biomarkers production and pathway activation. Since the health promoting properties of wine in different pathological conditions may include the reduction of inflammation, the aim of this paper was to collect and review the in vitro, in vivo, and human studies performed to evaluate the effects of wine on different models of inflammation. Although great variability in wine intake, period of consumption, and content of phenolic compounds was observed, data from both human and animal studies showed a positive modulation of inflammatory biomarkers (cytokines, coagulation parameters) and oxidative stress (mainly malondialdehyde) involved in cardiovascular function. In addition, some convincing evidence was obtained in different models suggesting a positive modulation of risk factors for gastric and intestinal inflammation. Contradictory results were obtained for metabolic syndrome and type 2 diabetes. To date, no significant paper has been published in the area of immune function. Integrating in vivo data and in vitro studies, the NF-κB pathway has been identified as a critical target for the protective properties of a moderate wine consumption.


Introduction
The interest in a positive role of wine on human health has grown since 1979, when a first report showing an inverse association between moderate wine consumption and mortality was published [1].This correlation has been confirmed by epidemiological observations; in particular, the lower cardiovascular mortality rate in France in comparison with other countries where a similar consumption of saturated fats was reported, the so-called "French paradox" [2].Since then, many authors have associated the consumption of wine with a J-shaped reduction of the risk factors for cardiovascular disease and certain cancers, as well as for diabetes and dementia [3,4].Although different factors are involved (age, phenotypes, dietetic habits, etc.), the observed beneficial effects of red wine in particular, are generally associated with the moderate consumption, which could be defined as consumption of 200-300 mL/day for men (corresponding to 20-30 g ethanol) and 100-200 mL/day for women (10-20 g ethanol) with meals [5,6].
Most scientific studies have suggested that any positive effects of wine on human health are associated with its phenolic compounds and ethanol, that can synergistically affect several biochemical factors with potential cardioprotective properties, including lipid profile, platelet aggregation, and endothelial function.In more detail, flavonoids and their metabolites, which are the most important phenolic fractions for human health, are generally responsible for the antioxidant and radical scavenging activity of wine.Red wine contains flavanols (500-1500 mg/L), flavonols (50-200 mg/L), and anthocyanins (90-400 mg/L) [7].In addition, a non-flavonoid phenolic compound has been extensively studied for its healthy potential: the stilbene monomer trans-resveratrol [8,9].According to some authors, the mechanism associated with the beneficial effects of wine could be due not only to phenolic compounds but also to an increase of uric acid concentration in plasma, and of its related antioxidant activity [10].If oxidative stress is recognized as one of the main factors in promoting different pathological conditions, an increasing body of evidence shows that several chronic diseases are modulated by inflammation; among others: arthritis, cancer, cardiovascular diseases, diabetes, chronic inflammatory bowel diseases (IBD), and neurological diseases [11].The dysregulation of various signaling pathways, such as the nuclear factor kappa-B (NF-κB) and signal transducer and activator of transcription 3 (STAT3), seems to be involved in inflammation.In fact, these factors regulate the transcription of specific enzymes, such as cycloxigenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-1, -6, -8, (IL-1, IL-6, IL-8), and chemokines) [12].The relationship between inflammation and chronic diseases has been suggested by different papers [11,13], as well as the possible positive role of wine in this process by in vitro and in vivo models.The results of these studies are often contradictory due to: (1) differences in the experimental model used; (2) heterogeneous biomarkers considered, since inflammation is a complex phenomenon, which can affect several physiological systems and pathways; (3) difficulty in selecting reliable biomarkers to measure the positive effects; and (4) the absence of the chemical characterization of wine, where several molecules (including alcohol) can be responsible for the increase or reduction of inflammatory biomarkers.
On the basis of the limits reported above, the aim of this paper was the collection and critical assessment of data present in the scientific literature, where inflammation was correlated with the intake of wine.In vitro and in vivo models as well as human trials will be considered, in order to identify the most suitable inflammatory biomarkers, and the most useful models/methods for the quantification of these biomarkers and their variations from moderate wine consumption.

Methods
The following databases were searched electronically to identify relevant articles published from database inception up to September 2018: PubMed/Medline, Embase, Cab Abstract.The search limits were in vitro, in vivo, clinical methodologies, and the European languages.A search strategy was developed for each database.using specific medical subject heading (MeSH) terms (e.g., inflammation mediators, inflammatory diseases, and wine) in addition to relevant text keywords (alcohol, wine, methods, analytical approaches).
Titles and abstracts of collected citations were first screened to identify the suitable publications, that is in vivo methods developed in humans or animals, and in vitro approaches used to evaluate the effect of wine/wine extracts in inflammatory pathways.Papers reporting data on the use of pure standard compounds, reviews, and patents were excluded.

Results and Discussion
The search by title and abstract retrieved 424 papers.After removal of duplicates and application of the inclusion/exclusion criteria, the total number of papers was 27.

In Vitro Methods
Table 1 reports the in vitro methods applied to investigate the effects of wine on parameters related to inflammation.Intestinal inflammation was the most common condition investigated, using Caco-2 and HT29 cells.Both are well characterized epithelial cell lines, derived from a primary colon tumor; thanks to their similarities with enterocytes of the human small intestine, they are a suitable model to study effects/absorption of food substances [12][13][14][15].Nicod and co-workers [15] and Nunes and co-workers [16,17] evaluated the effects of wine polyphenols on inflammatory biomarkers production by CaCo-2 and HT-29 cells, respectively.IL-6, IL-8, and COX-2 were significantly reduced in a concentration-dependent manner by the wine extracts.Different mechanisms of action were suggested by the authors [15][16][17].Nicod et al. speculated that microbiota modulation could be involved in the biological effects observed, since the expression of selected genes encoding for inflammatory mediators (IL-6, IL-8, ICAM, COX 2) was not affected.Nunes et al. [16,17] showed that wine polyphenols inhibited the NF-κB cascade activation, responsible for the expression of several inflammatory cytokines, such as IL-8 and COX-2, factors playing an important role in inflammatory bowel diseases (IBD).A further mechanism could be responsible for the positive effects of wine: it is the JAK/STAT and Nrf2 (Nuclear Factor erythroid 2-related factor) signaling pathways activated during inflammatory condition in IBD.These results indicate that polyphenols from a red wine extract (RWE) could act on multiple targets.According to the authors and previous studies [19], anthocyanins, the major compounds in RWEs, could be the most important actor, although the contribution of other polyphenols cannot be excluded.
A different 'wine' extract from Lenoir grapes (Vitis aestivalis hybrid) was used by Angel-Morales et al. [18] to investigate the effects of wine polyphenols on human colon-derived CCD-18Co myofibroblast cells, which are involved in T-cell function modulation in IBD.In these pathological conditions several genes encoding for inflammatory mediators (granulocyte-macrophage colony-stimulating factor, interleukins IL-a, IL-b IL-6, IL-8) and adhesion molecules (intracellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1)) are overexpressed in fibroblasts, as a consequence of inflammatory stimuli.This study focused on microRNAs (miRs), and in particular miR-126.MiRs are a class of endogenous noncoding RNAs, which are involved in post-transcriptional suppression of genes; miR-126 inhibits the expression of endothelial adhesion molecules reducing inflammation and, as a consequence, are an alternative target for the anti-inflammatory properties of natural components.
The induction of miR-126 by RWE (at the highest concentration, 100 µg/mL) was the mechanism by which the extract decreased the expression of VCAM-1.Although the extract was not obtained from Vitis vinifera, the results suggest the potential role of miR-126 in the beneficial properties of red wine in a model of intestinal inflammation.For this reason, further studies should be conducted to investigate if a similar effect can be obtained from Vitis vinifera derivatives, including wine.
In a model of endothelial inflammation, Calabriso et al. [14] showed that endothelial adhesion molecules (e.g., VCAM-1, ICAM-1, monocyte chemoactractant protein-1 (MCP-1), and E-selectin) were reduced by the lowest concentration of wine polyphenols tested (1 µg/mL), through a down-regulation of mRNA levels.This effect was mediated by the inhibition of NF-κB DNA binding activity exerted by coumaric acid, kaempferol, and resveratrol.Unfortunately, the effects of wine anthocyanins were not assessed, making a comparison with the other in vitro studies impossible.
The animal studies were performed in mice or rats, which are considered a good model for nutritional and pharmacological trials, also considering the short time required to observe significant changes in biochemical parameters after treatment.The length of studies varied from 14 days to 6 months.
The moderate consumption of red wine has been associated with a broad spectrum of health-promoting effects, but the presence of ethanol in different amounts, raised concerns as to whether or not the polyphenol content can exert positive effects beyond the ethanol toxicity.
The anti-inflammatory activity of red wine was also evaluated in different models of inflammation/oxidative stress induced by a high fat or fructose diet [21,26], or granuloma [20].Red wine significantly reduced some parameters of oxidative stress, such as 8-hydroxy-2'deoxyguanosine (8-OHdG) and malondialdehyde (MDA), biomarkers of DNA and lipid oxidation, respectively.In addition, Lopez and co-workers [20], investigating the possible differences between dealcoholized red (DRW) and white (DWW) wines, showed a greater antioxidant activity of DWR in vitro and in vivo models.This was probably due to the higher content of polyphenol, ascorbic acid and sulfites, and to the contribution of wine polyphenols in producing uric acid [10].According to Vazquez-Prieto and co-workers [26], the effects of the red wine can be attributed to the polyphenol content (total phenol content: 2.9 g/L gallic acid equivalents); in particular, catechin (24 mg/L) and resveratrol (1.1 mg/L) could be involved in the reduction of fat oxidation and the inhibition of preadipocyte proliferation.
The evaluation of oxidative stress was investigated, as secondary end-point, in several other studies since it is involved in the development of several disorders, including cardiovascular diseases.
Since data on biological effects of white wine are quite scarce in comparison with red wine, Rezic-Munizic et al. [22] and Kljucevic et al. [23], evaluated the effects of white wine on inflammatory biomarkers after myocardial infarction in rats.When compared to controls, wine consumption was associated with the increase in CD44 (cluster of differentiation 44) expression (0.2-fold, p < 0.0203), which has a role in fibrous tissue deposition in the healing infarct and in cardiac regeneration.White wine is relatively rich in non-flavonoid compounds, such as phenolic acids (derivatives of hydroxybenzoic and hydroxycinnamic acid) and other phenols such as tyrosol, that could mediate the effect on CD44 expression.A similar study was performed by Kljucevic et al. [23], who found a significant reduced expression of MMP-2 and MMP-9 (matrix metalloproteinases) in all the representative myocardial areas identified: infarct/ ischemic, peri-infarct/border zone, and control/non-ischemic zones (p < 0.001).This indicates a potential role of white wine in improving the inflammatory condition associated with cardiovascular impairment, since MMPs are responsible for the cleavage of several extracellular matrix proteins (activating cytokine production) and modulate the course and outcome of myocardial infarction.According to the authors' conclusions, however, this effect is not due to inhibition of the NF-κB pathway mediated by polyphenols.Unfortunately, neither of the two studies reported the chemical characterization of wine, making it difficult to establish a correlation between polyphenol content and the biological effects observed.
Finally, as in in vitro models, wine was evaluated in gastric and intestinal inflammation.Ethanol free RWE (5 mL/day) showed to be effective in reducing epithelial gastric damage induced in BALB/c mice by purified VacA (a vacuolar toxin produced by H. pylori), but not in influencing gastric colonization levels, probably because other mechanisms of infection by H. pylori are involved [25].Considering that gastritis may be caused by other factors, including lifestyle, further studies should be performed on the effect of wine on gastric mucosa, taking also into account the content of ethanol (one of the most important gastric inflammation inductor) and phenolic compounds.
In a murine model of colitis, a treatment with a dealcoholized RWE (500 mg/kg bw) from muscadine grape (Vitis rotundifolia) was able to significantly reduce fecal myeloperoxidase (MPO) expression (from 10.4 to 5.39 units/mg of tissue); MPO is a pro-oxidative and pro-inflammatory enzyme produced by neutrophils and monocytes, which accumulates in intestinal mucosa in ulcerative colitis.The authors speculated that this effect could be mediated by anthocyanins [27], the content of which was 171.2 mg/g, more than half of the total polyphenol amount present in the extract.

Human Studies
Table 3 describes the studies in humans performed to evaluate the effects of wine on inflammation, with indication of the models used.Twelve among the 15 trials were performed on healthy subjects.They were generally designed as randomized, controlled, and cross-over studies where the targets were the cardiovascular and immune function.To evaluate the effect of "acute" consumption on inflammation, wine intake ranged from 150 mL to 500 mL/day in a single dose; to simulate the "regular" intake the period was longer: from 15 days up to 12 months.When reported, total polyphenol content of wine varied from 1.5 to 3.0 g/L [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43].Among inflammation parameters investigated, C-reactive protein (CRP) was one of the most considered, since it has been identified by several authors as a significant predictor of cardiovascular events [44].Acute red wine intake (fasting or at meals) was not associated with significant C-reactive protein reduction, as observed by Cuervo et al. [28], and Retterstol et al. [29].This could be due to different factors; among the others: 1.
The duration of the study, which could be too short to observe a significant change in inflammatory parameters in healthy subjects having physiological CRP levels (0.11-0.55 mg/dL) [28].The study by Marfella et al. [42], seemed to support this hypothesis.Indeed, T2D patients, with a myocardial infarction history, consuming 118 mL wine/day in the previous 12 years showed CRP levels significantly lower when compared to abstainers (p < 0.01) [42]; 2.
The amount of wine consumed (100-150 mL/day, 15 g ethanol) was lower when compared to studies where a CRP reduction was observed (>200 mL, 20 g ethanol) [36].
Fibrinogen and other factors involved in platelet aggregation (e.g., platelet-activating factor-PAF; von Willebrand factor-vWf; plasminogen activator inhibitor-PAI-I; plasminogen activator inhibitor/tissue plasminogen activator-PAI/tPA ratio) appear to be affected by both acute and regular intake of both red and white wine.The acute effect of ethanol on platelet aggregation is controversial, as both inhibition and stimulation effects have been reported [45,46].In the studies described in this review, red wine, but not ethanol, water or white wine alone, reduced platelet aggregation within 3.5 h, suggesting an additional positive effect of polyphenols of red wine.Two studies reported an additional effect on circulating endothelial adhesion molecules ICAM-1 and VCAM-1, after red wine consumption (160-280 mL/day) [36,43].If confirmed, these effects could explain the reduced vWf levels observed in moderate wine diabetic consumers, since hyperglycemia stimulates platelet aggregation [43].Taking into account the number of studies collected and the low number of trial participants, however, the effects of wine on aggregation has to be investigated in further well-designed studies.
As regards other inflammatory parameters, such as interleukins and their gene expression, some discrepancies have been observed in the literature.Schwarz et al. [37] and Sacanella et al. [34] observed a significant reduction of IL-6 levels and its expression, after acute or regular wine intake (4 weeks 200 mL red wine + 4 weeks 200 mL white wine) in healthy subjects.On the other hand, no effects on IL-6 were found by Argyrou et al. [32] and Tousolis et al. [33] after acute consumption of wine.This result was confirmed by Williams et al. [38], who measured an IL-6 increase in patients with CAD (coronary artery disease).It is possible that in acute consumption, the alcohol-mediated oxidative stress in the liver may be involved in the cytokines production increase [47]; on the other hand, longer term wine consumption could enable the wine phenolic fraction to counteract this imbalance.In fact, according to in vitro studies, flavonoids in wine suppress the synthesis of proinflammatory cytokines and chemokines (such as TNF-α, IL-1b, IL-6, IL-8, and MCP-1, von Willebrand plasma levels), mainly via NF-κB pathway modulation [45].
Neither regular (500 mL/day for 2 weeks) nor acute (200 mL/one dose) consumption of red wine were associated with a positive effect on inflammatory biomarkers associated with immunological responses such as neutrophils and monocytes phagocytic activity and T cell apoptosis in healthy subjects [39,40], confirming that NF-κB is a possible mediator of the wine protective effects.
In in vitro models, wine extracts were generally used in an amount aimed to mimic the in vivo polyphenol concentrations observed in the colon [48].The reduction of inflammatory cytokines (e.g., IL-6, IL-8, COX-2, and VCAM-1) was observed using red wine polyphenol concentrations in the range of 0.17-600 µg/mL.These effects seem to be mediated by the inhibition of NF-κB DNA binding activity, while other pathways seem to be less involved (i.e., AP-1).According to the authors and previous studies [19] anthocyanins, which are among the most abundant compounds in RWEs, could be the main responsible for the biological effects observed, although the contribution of other polyphenols, including procyanidins, cannot be excluded.Moreover, coumaric acid, kaempferol, and resveratrol showed a lower concentration but a specific higher activity in reducing the inflammatory parameters.
In animal studies, MDA and 8-OHdG, markers of oxidative stress, were significantly reduced by wine.It should be noticed that in most studies, dealcoholized extracts were used, so that only the effect of wine polyphenols was evaluated.In gastric/intestinal areas, animal studies showed promising positive effects for wine (50-250 mL/kg bw rats or mice) in reducing cytokine production and gastritis severity associated with H. pylori infection.This effect has to be confirmed, however, in human studies.

Conclusions
The purpose of this review was to collect and discuss the experimental approaches and the scientific evidences of in vitro and in vivo studies aimed to evaluate the relationship between wine consumption and inflammation.Even though moderate wine consumption has been associated with lower risk for cardiovascular diseases, a great heterogeneity characterizes the studies performed to establish a correlation among beneficial effects of wine consumption: wine typology, composition (when reported), biomarkers measured, length and amount of consumption, together with the use of appropriate controls make difficult a suitable comparison.
In humans, inflammatory parameters associated with platelet aggregation seem to be affected by moderate wine intake, while contradictory results were obtained for some inflammation biomarkers (e.g., CRP and IL-6).Conversely, no significant effects were measured in inflammatory responses associated with immune function.For this reason, further studies (including in vitro models), evaluating wine effects on biomarkers of immune response, should be designed.Integration of in vitro studies with animal and human trials could lead to a better wine effects evaluation and comprehension of the most plausible mechanisms of action.

Table 1 .
Selected papers describing in vitro trials describing the wine effects on inflammation.

Table 2 .
Summary of the collected animal studies used to assess effects of wine on inflammation biomarkers.

Table 3 .
Summary of the collected human trials to assess effects of wine on inflammation biomarkers

Table 4 .
Summary of in vitro, animal, and human studies reviewed.