In vitro assessment of the ability of probiotics, blueberry and food carbohydrates to prevent S. pyogenes adhesion on pharyngeal epithelium and modulate immune responses

Valentina Taverniti a, Alessandro Dalla Via a, Mario Minuzzo b, Cristian Del Bo’ a, Patrizia Riso a, Hanne Frøkiær c and Simone Guglielmetti *a
aDepartment of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Italy. E-mail:
bDepartment of Biosciences, University of Milan, Italy
cDepartment of Veterinary and Companion Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

Received 7th June 2017 , Accepted 26th August 2017

First published on 28th August 2017

Group A streptococci (GAS) cause 20–30% of pediatric pharyngitis episodes, which are a major cause of ambulatory care visits. Therefore, a strategy to prevent GAS dissemination in children could significantly benefit public healthcare. Contextually, we assessed the possibility of employing alternative food-grade strategies to be used with the oral probiotic L. helveticus MIMLh5 for the prevention of pharyngeal infections. First, we demonstrated through an antagonism-by-exclusion assay that guaran may potentially prevent S. pyogenes adhesion on pharyngeal cells. Subsequently, we showed that an anthocyanin-rich fraction extracted from wild blueberry (BbE) exerts anti-inflammatory effects on the human macrophage cell line U937. Finally, we showed that BbE reduces interferon-β expression in MIMLh5-stimulated murine dendritic cells, resulting in a reduction in the pro-inflammatory cytokines IL-12 and TNF-α. In conclusion, this proof-of-concept study indicates that different food-grade strategies may be concomitantly adopted to potentially prevent GAS colonization and modulate local immune defences.

1. Introduction

The nasal and oral cavities are the main gates of entrance for microorganisms from the external environment. The primary site for potential colonization is then the pharyngeal mucosa, which is in fact a frequent location for bacterial and viral infections. To manage such risk, hosts have developed a complex system of immunological defenses, which are collectively known as the nasal-associated lymphoid tissue or the Waldeyer's ring (WR). The WR is mainly composed of the lingual tonsil, adenoid, and palatine tonsils. The epithelium of the palatine tonsils extends into a complicated network of invaginations, making up crypts where molecules and microorganisms constantly diffusing from the oropharyngeal cavity meet a rich and abundant set of immune cells, such as monocytes, macrophages and dendritic cells, which respond to stimuli, affecting both local and systemic immunity.1

Streptococcus pyogenes is a beta-hemolytic bacterium of the Lancefield serogroup A, which can efficiently elude the immune barrier of the WR. S. pyogenes may be the cause of a wide range of pathological conditions, ranging from local (e.g., pharyngitis2) to invasive systemic (e.g. endocarditis3) infections. Notably, S. pyogenes is the most common bacterial etiological agent of acute pharyngeal infections, especially in school-age children,4,5 and it is, consequently, a primary reason for antibiotic prescription in the pediatric population.6

The use of live microorganisms for the prevention and treatment of pharyngeal infections (i.e., the probiotic approach) has been investigated, leading to the development of probiotic products, which are already available on the market. The most successful example is the BLIS range of products based on Streptococcus salivarius K12, a bacterium originally isolated from the oral cavity of a healthy child, which have been demonstrated in several studies to colonize the oral cavity,7 inhibit pathogenic streptococci through the production of the lantibiotic bacteriocin salivaricin,8 and modulate immune responses.9 Another bacterium that has been selected as a probiotic candidate for use in the pharyngeal mucosa is the dairy strain Lactobacillus helveticus MIMLh5, which possesses the ability to antagonize S. pyogenes10 and to modulate the immune response in epithelial cells,10 macrophages11 and dendritic cells12 also, independently from its viability, due to the presence of an abundant immune-active surface layer (S-layer) protein on the outer surface of the bacterium.12

In this study, we assessed the possibility of using alternative food-grade strategies for the prevention of pharyngeal infections, which can be adopted concomitantly with the use of the oral probiotic L. helveticus MIMLh5. Specifically, since S. pyogenes adhesion on epithelial cells in vivo is mediated by its capsule polysaccharide hyaluronate,13 here we tested the ability of several food carbohydrates (i.e., alginate, aloe, arabinogalactan, chitosan, guar gum, inulin, pectin, starch and xylan) to inhibit the adherence of this pathogen in vitro to FaDu pharyngeal cells. In addition, we investigated an anthocyanin (ACN)-rich wild blueberry extract, since this food component was demonstrated to exert anti-inflammatory properties on epithelial cells14 and, therefore, could contribute to the management of inflammation occurring during pharyngeal infections.

2. Materials and methods

2.1 Food carbohydrates, bacterial strains and blueberry extract

All the carbohydrates used in antagonism experiments on FaDu cells were from Sigma-Aldrich (Steinheim, Germany) except for the aloe extract, which was from ESI (Aloe Vera Esi Gel 99.9%; ESI S.p.A., Albissola Marina, Italy). In the study, we used Streptococcus pyogenes C11, belonging to emm-type 77, which was originally isolated from a pharyngitis patient. S. pyogenes was grown in Brain Heart Infusion broth (BHI; Difco, Detroit, USA) supplemented with 0.3% yeast extract at 37 °C for 18–24 h. For the cultivation of the recombinant S. pyogenes strain C11LucFF,15 5 μg ml−1 chloramphenicol was added to the medium. Lactobacillus helveticus MIMLh5 (from the food microbiology culture collection of the Department of Food, Environmental and Nutritional Sciences, University of Milan) was cultivated in de Man, Rogosa and Sharpe medium (MRS; Difco) at 37 °C, overnight. The blueberry extract (BbE) used in the study was prepared as previously described,14 using a freeze-dried, powdered, Vaccinium angustifolium berry preparation standardized at 1.5% total ACNs from FutureCeuticals (Momence, IL, USA).16,17 Specifically, the BbE corresponds to the ACN-fraction obtained and characterized previously by Taverniti et al.14 The ACN-fraction was dissolved and stabilized in methanol acidified with 0.05 mM HCl (MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl). The BbE contained 14 different ACNs, with malvidin and delphinidin glycosides as dominant compounds, followed by cyanidin, petunidin and peonidin glycosides.14

2.2 Experiments on the FaDu cell layer

The human pharynx carcinoma cell line FaDu (from the American Type Culture Collection, ATCC HTB-43) was cultivated at 37 °C under an atmosphere of 95% air and 5% carbon dioxide, until a confluent monolayer was formed. The culture broth was Dulbecco's Modified Eagle's Medium (DMEM; Lonza, Basel, Switzerland), supplemented with 10% (v/v) heat-inactivated (at 56 °C for 30 min) fetal calf serum, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 0.1 mM non-essential amino acids, and 2 mM L-glutamine (Sigma-Aldrich).

The antagonism-by-exclusion assay was performed with the recombinant bioluminescent reporter strain S. pyogenes C11LucFF, which was previously developed through the introduction of the reporter vector pCSS945, carrying a phage T5 promoter-lac operator upstream of the insect luciferase gene lucGR.15 In brief, the experiment consisted of the 1-hour pre-incubation of the FaDu layer with 10 g l−1 of the carbohydrates under study, followed by a washing step with PBS (pH 7.3) and 1-hour incubation with approximately 2 × 108S. pyogenes C11LucFF cells per ml resuspended in PBS (the bacterial concentration was determined microscopically by means of a Neubauer improved counting chamber, Paul Marienfeld GmbH & Co. KG). After two washes with PBS, D-luciferin (Sigma-Aldrich) was added at a concentration of 12.5 μM (in citrate buffer, pH 5) and the luminescence signal was immediately measured with a Victor 3 luminometer (PerkinElmer, Monza, Italy).

For bacterial adhesion experiments, FaDu cells were grown as defined above on microscope cover glass, following a previously described method.18 In brief, an FaDu layer was incubated for 1 h in PBS supplemented with 10 g l−1 of the following carbohydrates: alginate, aloe, arabinogalactan, chitosan, guar gum, inulin, pectin, starch and xylan. In addition, hyaluronate, which constitutes the exopolysaccharide capsule of S. pyogenes, was been used as a positive control (10 g l−1). Afterwards, the cell layer was gently washed (three times) with PBS and incubated for 1 h with approximately 2 × 108 cells of an overnight culture of S. pyogenes resuspended in PBS. Adherent bacteria and FaDu cells in 10 randomly selected microscopic fields were counted and averaged; results were expressed as an adhesion index, i.e., the number of bacterial cells per 100 FaDu cells.

2.3 Experiments with the U937 cell line

U937 cells (from the American Type Culture collection, ATCC CRL-1593.2) were seeded at a density of 5 × 105 cells per well in 24-well plates and cultivated at 37 °C under a 5% CO2 humidified atmosphere in RPMI 1640 medium (Lonza) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco-BRL, Life Technologies, Milan. Italy), 2 mM L-glutamine, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin (Sigma-Aldrich). Differentiation into adherent, non-replicative macrophage-like cells19,20 was induced for 48 h through the addition of phorbol myristate acetate (PMA; Sigma-Aldrich) into the cellular medium at a final concentration of 0.1 mM. Afterwards, cells were washed once with sterile PBS buffer to remove all non-adherent cells. One hour before stimulation, the culture medium was replaced with RPMI 1640 supplemented with 1% (v/v) FBS to enable cell adaptation. Lipopolysaccharide (LPS, final concentration 1 μg ml−1) from Escherichia coli 0127:B8 (Sigma-Aldrich) was used as a pro-inflammatory stimulus. BbE was tested at three different concentrations (1, 10 and 25 μg ml−1). U937 cells were also treated with MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl to evaluate possible interference from the BbE solvent on U937 responses. Under all tested conditions, the final concentration of MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl was lower than 0.1% (v/v). Two different experimental set-ups were used: 1 h of pre-incubation with LPS followed by BbE supplementation for 3 h, and 1 h of pre-incubation with BbE followed by treatment with LPS for 3 h.

After incubating macrophages for a total of 4 h (1 h pre-incubation + 3 h treatment), according to the two experimental set-ups described above, the supernatant was carefully removed from each well and the total cellular RNA was isolated from the adhered cells and converted to cDNA, as previously described.11 The expression levels of TNF-α gene were then analyzed via quantitative PCR, using SsoFast EvaGreen Supermix (Bio-Rad Italia, Segrate, Italy) on a Bio-Rad CFX96 system, as previously described.11,12 Reference gene coding for glyceraldheyde-3-phosphate dehydrogenase was used to normalize gene expression. Gene expression levels are reported as the fold of induction (FOI) in comparison with the control (namely, unstimulated U937 cells), to which we attributed a FOI value of 1.

2.4 Experiments with murine bone marrow derived dendritic cells (BMDCs)

All animals used as a source of bone marrow cells were housed under conditions approved by the Danish Animal Experiments Inspectorate (Forsøgdyrstilsynet), Ministry of Justice, Denmark, and experiments were carried out in accordance with the guidelines ‘The Council of Europe Convention European Treaty Series 123 for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes’. Since the animals were employed as sources of cells, and no live animals were used in experiments, no specific approval was required for this study. Hence, the animals used for this study are included in the general facility approval for the faculty of Health and Medical Sciences, University of Copenhagen. BMDCs were prepared as described previously.21 Briefly, bone marrow from C57BL/6 mice (Tactonic, Lille Skensved, Denmark) was flushed out from the femur and tibia and washed. Bone marrow cells (3 × 105) were seeded into 10 cm Petri dishes in 10 ml of RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) containing 10% (v/v) heat inactivated fetal calf serum supplemented with penicillin (100 U ml−1), streptomycin (100 mg ml−1), glutamine (4 mM), 50 mM 2-mercaptoethanol (all from Cambrex Bio Whittaker) and 15 ng ml−1 murine GM-CSF (harvested from a GM-CSF transfected Ag8.653 myeloma cell line). The cells were incubated for 8 days at 37 °C under a 5% CO2 humidified atmosphere. On day 3, 10 ml of complete medium containing 15 ng ml−1 GM-CSF was added. On day 6, 10 ml was removed and replaced with fresh medium. Non-adherent, immature DCs were harvested on day 8. Afterwards, immature DCs (2 × 106 cells per ml) were resuspended in fresh medium supplemented with 10 ng ml−1 GM-CSF, and were seeded at a concentration of 500 μl per well in 48-well tissue culture plates (Nunc, Roskilde, Denmark).

To prepare fresh cultures to be used in immunological experiments, bacterial cells from an overnight culture were collected, washed twice with sterile PBS, counted with a Neubauer improved counting chamber, and resuspended at a known concentration in the same medium used to culture the BMDCs. L. helveticus MIMLh5 was tested at a multiplicity of infection (MOI) of 5. In co-incubation experiments, BbE was used at concentrations of 1, 3 and 10 μg ml−1, and was added to the BMDCs 30 min before the addition of L. helveticus. When tested alone, BbE was used at concentrations of 5, 25 and 50 μg ml−1. Cell supernatants were harvested 20 h after bacterial stimulation and tested for cytokine concentration via sandwich ELISA duosets (RnD Systems, Abingdon, UK), according to the manufacturer protocol. For the gene expression analysis, BMDCs were harvested for RNA extraction after 4 h. The total RNA was extracted using the MagMAX sample separation system (Applied Biosystems, Foster City, CA), including a DNAse treatment step for genomic DNA removal. RNA concentration was determined via Nanodrop analysis (Thermo, Wilmington, DE). Then, 500 ng of the total RNA was reverse transcribed using the TaqMan Reverse Transcription Reagent kit (Applied Biosystems, Foster City, USA) using random hexamer primers, according to the manufacturer instructions. The thermic cycle used for retrotranscription was 10 min at 25 °C, 120 min at 37 °C, and 5 s at 85 °C. The obtained cDNA was stored in aliquots at −80 °C. Primers and probes were obtained as previously described.22 The probes were labelled with the 5′ reporter dye 6-carboxy-fluorescein (FAM) and the 3′ quencher dye NFQ-MGB (Applied Biosystems). The amplifications were carried out in a total volume of 10 μl, containing 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), forward and reverse primer, TaqMan MGB probe and purified target cDNA (6 ng in the reaction). The cycling parameters were initiated for 20 s at 95 °C, followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C, using the ABI Prism 7500 (Applied Biosystems). Amplification reactions were performed in triplicate, and DNA contamination controls were included. The amplifications were normalized to the expression of the β-actin encoding gene. Relative transcript levels were calculated by applying the 2(−ΔΔCT) method. Data were collected from three independent experiments carried out in duplicate.

2.5 Statistical analysis

The experiments were carried out in duplicate at least twice. An unpaired Student's t-test or one-way analysis of variance (ANOVA) with Dunnet's post test were run for statistically significant differences.

3. Results

3.1 S. pyogenes adhesion to the FaDu hypopharyngeal carcinoma cell layer may be inhibited by food carbohydrates

The ability of nine different carbohydrates to inhibit S. pyogenes adhesion was studied by means of an antagonism-by-exclusion assay based on a three-component system, consisting of the FaDu cell layer, the bioluminescent mutant S. pyogenes C11LucFF, and the selected carbohydrate molecule. In this experiment, reduction in bioluminescence was measured to assess the potential ability of the carbohydrates to prevent the epithelial layer being colonized by the pathogen. All carbohydrates were tested at 10 g l−1, since this concentration was found during the set-up of the experiment to obtain about an 80% reduction in luminescence using hyaluronate (the polysaccharide that mediates S. pyogenes adhesion to epithelial cells13), ensuring its appropriateness as a positive control. Besides hyaluronate, four other carbohydrates significantly modified the bioluminescence emitted by S. pyogenes; specifically, xylan and chitosan induced a significant increase, whereas guar and aloe gel produced a significant reduction in the relative luminescence units (RLU) of S. pyogenes C11LucFF (Fig. 1A).
image file: c7fo00829e-f1.tif
Fig. 1 The effect of food carbohydrates on S. pyogenes colonization of an FaDu hypopharyngeal epithelial cell layer. A. The antagonistic exclusion activity of carbohydrates against bioluminescent Streptococcus pyogenes C11lucFF on FaDu cells. Data are reported as percentage variations in light emission (RLU, relative luminescence units), as compared to a cell layer treated with only PBS (control) before incubation with S. pyogenes. Data are represented as Tukey boxes and whiskers, and derive from at least three independent experiments performed in duplicate. B. The adhesion of S. pyogenes C11 to an FaDu cell layer after treatment of the cell layer with carbohydrates. Data are expressed as adhesion indices (i.e., the number of adhered bacteria per 100 FaDu cells), and represent the mean (+ standard deviation) of at least two independent experiments conducted in duplicate. C. S. pyogenes C11 adhesion to FaDu cell monolayers as observed with Giemsa staining under a light microscope at 1000× magnification. Statistically significant differences compared to a control are according to an unpaired Student's t-test (*** P < 0.001; * P < 0.05).

Subsequently, to verify whether the ability of guar and aloe gel to inhibit bioluminescence was associated with the reduced adhesion of S. pyogenes to the FaDu cell layer, we performed adhesion experiments. The analysis was carried out by counting with an optical microscope the number of adhered S. pyogenes cells after the incubation of the FaDu layer with the carbohydrate. Specifically, we tested the two potential inhibitory carbohydrates identified in the previous experiment (i.e., guar and aloe gel), a low-viscosity oligosaccharide (i.e., inulin) and a high-viscosity polysaccharide (i.e., alginate). We found that, in addition to the positive control hyaluronate, only guar significantly inhibited S. pyogenes adhesion. On the contrary, the reduction in pathogen adhesion using aloe gel, although demonstrated to drastically reduce bioluminescence, was not significant (Fig. 1B and C). In a separate set of adhesion experiments, we also ascertained that blueberry extract used alone or in combination with guar did not affect the adhesion of S. pyogenes on FaDu cells (data not shown).

Overall, these results suggest that guar gum may potentially prevent pharyngeal epithelial colonization by the pathogen S. pyogenes.

3.2 Blueberry extract possesses anti-inflammatory properties on U937 human macrophages

In the subsequent part of the study, we investigated the potential ability of the ACN-rich fraction from freeze-dried wild blueberry to prevent an inflammatory response. For this aim, we used an immunological model based on PMA-differentiated U937 human macrophages, stimulated with E. coli LPS molecules to induce a pro-inflammatory response. The presence of MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl, which was used to solubilize the blueberry extract (BbE), had only a slight effect on the TNF-α expression level induced by LPS (Fig. 2). Specifically, BbE was added to U937 cells before or after treatment with the inflammatory stimulus LPS. We found that the highest concentration of BbE tested (25 μg ml−1) reduced LPS-induced induction of TNF-α gene expression; however, statistical significance was reached only when U937 cells were pre-stimulated with BbE (p < 0.05), whereas only a trend towards a reduction in inflammatory induction (p = 0.085) was observed when U937 cells were treated with BbE after LPS stimulation (Fig. 2).
image file: c7fo00829e-f2.tif
Fig. 2 The effect of blueberry extract on U937 macrophages. A quantitative analysis of TNF-α gene expression in U937 cells stimulated for 4 h with blueberry extract (BbE). Expression levels of the TNF-α gene are indicated through fold of induction (FOI) relative to a control (unstimulated U937 cells), which was set at a value of 1. LPS was used at a concentration of 1 μg ml−1. LPS + Met[thin space (1/6-em)]:[thin space (1/6-em)]HCl refers to U937 cells stimulated with LPS supplemented with methanol + 0.05 mM HCl (MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl) at a concentration corresponding to the concentration used for BbE samples. BbE was tested at concentrations of 25, 10 and 1 μg ml−1. MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl was added to all samples to reach the concentration present in the highest quantity of BbE used. 1st stimulation refers to the 1 h pre-stimulation of U937 cells; 2nd stimulation refers to the 3 h stimulation of U937 cells. Data from at least three independent experiments are reported as a mean (+ standard deviation). Horizontal square brackets indicate statistically significant differences (*, P < 0.05; **, P < 0.01) according to an unpaired Student's t-test; p values between 0.05 and 0.1 are also indicated.

Overall, these results indicate that the ACN-rich fraction extracted from freeze-dried wild blueberry (i.e. BbE) exerts anti-inflammatory activity on macrophages in vitro.

3.3 Blueberry extract affects the effects of L. helveticus MIMLh5 on dendritic cells

In the last set of experiments, we tested the immunomodulatory properties of BbE on BMDCs in the presence of the immune-active probiotic strain L. helveticus MIMLh5. Specifically, BMDCs were co-stimulated with MIMLh5 cells (MOI 5) and increasing concentrations of BbE, and the resulting cytokine production was assessed. MIMLh5 alone resulted in 3.5 ng ml−1 TNF-α and 250 pg ml−1 IL-12. MIMLh5 alone or in combination with BbE did not induce proinflammatory IL-1β in the BMDCs (data not shown). The addition of BbE determined a significant reduction in both TNF-α and IL-12 at the highest tested concentrations (Fig. 3A). Particularly, we observed a strong reduction in the MIMLh5-induced production of the pro-inflammatory cytokine IL-12 (about −89%) when 50 μg ml−1 BbE was used (Fig. 3A).
image file: c7fo00829e-f3.tif
Fig. 3 The modulation of cytokine production in murine bone marrow derived dendritic cells. A. Murine bone marrow derived dendritic cells (BMDCs) stimulated for 30 min with BbE before the addition of media or Lactobacillus helveticus cells at a multiplicity of infection (MOI) of 5, with subsequent 20 h stimulation and harvesting of cell supernatant. BbE was tested at concentrations of 0, 1, 10 and 50 μg ml−1. Methanol + 0.05 mM HCl (MetOH[thin space (1/6-em)]:[thin space (1/6-em)]HCl) was added to all samples to reach the concentration present in the highest quantity of BbE used. Cytokine concentrations were measured in the supernatant and expressed as relative values. B. BMDCs were harvested 4 h after the addition of bacteria and RNA was extracted for gene expression analysis via RT-qPCR. FOI: fold of induction. Data from three independent experiments conducted in duplicate are reported as a mean ± standard deviation. Asterisks indicate results from stimulation with MIMLh5 and BbE that are statistically different from cells only stimulated with MIMLh5 (*, P < 0.05; **, P < 0.01, ***, P < 0.001) according to one-way ANOVA with Dunnet's post test.

It was previously shown that upon stimulation of BMDCs with various lactobacilli, IL-12 can be primarily induced indirectly through the transient up-regulation of IFN-β around 4–6 h after the addition of bacteria, which in turn stimulates the induction of IL-12 and to a lesser extend TNF-α through binding to the IFN activating receptor (IFNAR).23 To study how the addition of blueberry extract affected the IL-12 inducing pathway, we harvested cells after 4 h of stimulation and extracted RNA for RT-PCR analysis of Il12 and Infβ expression. After 4 h of stimulation, the expressions of both Ifnβ and Il12 were affected only by the addition of 50 μg ml−1 BbE (Fig. 3B).

Overall, these experiments demonstrate that the ACN-rich fraction extracted from freeze-dried wild blueberry reduces the expression of the cytokine IFN-β in murine bone marrow-derived dendritic cells that have been stimulated by L. helveticus MIMLh5, and that this in turn leads to a reduction in the pro-inflammatory cytokines IL-12 and TNF-α.

4. Discussion

Pharyngitis is a major cause of pediatric ambulatory care visits and often leads to antibiotic prescription.24 Up to 20% of children are carriers of group A streptococci (GAS),24 which are estimated to cause 20% to 30% of acute pharyngitis episodes in this population. Therefore, a strategy to prevent GAS dissemination in children could result in significant benefits for public health care. Contextually, in this study we tested the in vitro effectiveness of food-grade strategies that can be potentially used together to prevent pharyngeal infections and, specifically, GAS colonization of the oropharyngeal mucosa. Particularly, we focused on strategies that can be compatible with food applications, since food products may be consumed on a larger scale than pharmaceutical formulations.

The first step in the colonization of the oropharyngeal mucosa by Streptococcus pyogenes includes the recognition of the hyaluronate bacterial capsule by CD44 proteins exposed on the outer surface of oral epithelial cells.5 Accordingly, in vivo and in vitro experiments supported the potential efficacy of disrupting the interaction between the S. pyogenes hyaluronic acid capsule and CD44, to prevent pharyngeal infection.13 Contextually, the initial part of the present study was devoted to the selection of an edible carbohydrate possessing the ability to antagonize S. pyogenes colonization, similarly to hyaluronate. Out of nine different carbohydrates tested, only aloe gel and guar gum demonstrated significant inhibition of S. pyogenes colonization in vitro on the FaDu cell layer, assessed through determining intracellular ATP as a measure of bacterial cell wellbeing. Nonetheless, only guar could significantly inhibit S. pyogenes adhesion, indicating that, plausibly, the observed intracellular ATP reduction induced by aloe gel was due to toxic activity of the extract on the bacterial cells, rather than the preclusion of pathogen adhesion.

Guar gum, also called guaran, is a polysaccharide composed of the sugars galactose and mannose, which is obtained from maceration of the seeds of the plants Cyamopsis tetragonoloba (Linne) Taub. or Cyamopsis psoraloides (Lam.) D.C. Guar gum is an ingredient that meets the specifications of the “Food Chemicals Codex” (3rd edition, 1981, page 141), and has been generally recognized as safe (GRAS) by the U.S. Food and Drug Administration under the provisions of the Code of Federal Regulations (title 21 CFR 121.101). In Europe, the Food Safety Authority (EFSA) has stated that “there is no safety concern for the general population at the refined exposure assessment of guar gum (E 412) as a food additive”.25 In this study, we used a concentration of guar of 10 g per liter, which is compatible with its use in several food matrices, from bread to yogurt or fruit juice.26–28

Increasing evidence suggests the ability of plant polyphenols to affect immune regulation.29 For instance, we recently demonstrated the ability of an anthocyanin-rich extract from wild blueberry to exert anti-inflammatory activity on intestinal epithelial cells in vitro.14 Nonetheless, the ability of food polyphenols to modulate immune responses in vivo can be greatly hindered at the intestinal level due to limited diffusion through the mucin layer, reduced absorption, and rapid degradation by intestinal microorganisms. On the contrary, the same bioactive polyphenols may plausibly have greatly increased chances to interact with the immune system at the oral and pharyngeal levels, where immune cells, such as macrophages and dendritic cells, may come into direct contact with food molecules in structures specifically evolved for this purpose, such as the tonsillar crypts. The immune system associated with the oral cavity and the pharynx, in fact, is continuously challenged by antigens from air and food, and has the primary role of avoiding pathogen entry while preserving immune homeostasis.30 Therefore, plausibly, specific food components may activate and modulate immune responses occurring at the WR. Contextually, in this study we tested the ability of the same anthocyanin-rich blueberry extract previously used on intestinal epithelial cells14 to also demonstrate immunomodulatory properties toward antigen presenting cells. Specifically, here we used both macrophage and dendritic cell models, because macrophages are directly involved in the rapid resolution of local inflammation, whereas dendritic cells are specialized antigen presenting cells, which can convert a local response into a systemic effect by differently activating T cells populations depending on the stimuli they receive and process.31 Consequently, anthocyanin-rich blueberry extract (BbE) was used to stimulate PMA-treated U937 cells, which were employed as an in vitro model of macrophages. U937 cells were treated with BbE before the addition of the pro-inflammatory stimulus LPS, to investigate a possible preventive role towards an incoming inflammatory agent (inflammation-preventing experiment); in addition, the experiment was also performed by supplementing BbE after LPS stimulation, to assess the potential anti-inflammatory activity (inflammation-reducing experiment). The obtained results evidenced the ability of BbE to reduce the LPS-dependent expression of TNF-α, a cytokine induced within the NF-κB pathway that is involved in inflammatory responses;32 nonetheless, statistical significance was reached only in the inflammation-preventing experiment. Reportedly, anti-inflammatory properties have been shown for different categories of polyphenols. For instance, the pretreatment of BV-2 microglial cells in vitro with blueberry extract before LPS stimulation showed a concentration-dependent reduction in TNF-α release.33 Our results are also in accordance with those of Pergola et al.,34 who demonstrated that 2 h pre-treatment with an anthocyanin fraction from blackberry extract before LPS stimulation could inhibit iNOS protein expression in a murine J774 macrophage cell line. Furthermore, it was reported that supplementation with grape powder extract reduced the LPS-induced production of inflammatory cytokines in U937 macrophages,35 and a proanthocyanidin-rich cranberry fraction decreased LPS-induced cytoxicity in macrophages and oral epithelial cells.36 Finally, we recently documented that upon supplementation with an ACN-rich fraction, single ACNs and their gut metabolites were able to reduce lipid accumulation in THP-1 derived macrophages37 and counteract their adhesion to endothelial cells in a TNF-α stimulated pro-inflammatory environment.38

In this study, we also assessed the immunomodulatory potential of anthocyanin-rich BbE on mouse bone marrow-derived dendritic cells (DCs). DCs constitute a link between innate and adaptive immunity, since they act as professional antigen-presenting cells (APCs). This function is crucial in initiating an adaptive immune response, as T cells do not respond to free antigens but only to antigens that are presented by APCs. Several dendritic cell subsets have been identified in tonsils, where they can play different roles in regulating immunity or tolerance to antigenic material coming from the mouth and the nose.39 It has been proposed that DCs in the tonsils should mainly possess the default role of maintaining tolerance for or ignorance towards the numerous harmless food and microbial antigens coming from the nasopharynx.40 Exacerbated activation toward harmless stimuli would lead to the enhanced production of pro-inflammatory cytokines such as TNF-α and IL-12, leading to (low-grade) inflammation, which has been associated with susceptibility to infection.41,42 In this context, we can speculate that the observed properties of the blueberry preparation are potentially beneficial, since BbE does not trigger the production of pro-inflammatory cytokines by DCs and, notably, gives rise to a more balanced/less stimulatory cytokine profile in combination with MIMLh5, mitigating the stimulatory attitude of L. helveticus MIMLh5 toward TNF-α and IL-12 in DCs. L. helveticus MIMLh5 was included in this study since it is a bacterial strain that was selected in previous studies for its abilities to efficiently adhere to the FaDu epithelial cell line10,11 and antagonize S. pyogenes on FaDu and HaCat keratinocytes in in vitro models.10 In addition, this strain was shown to inhibit the IL1β-induced activation of NF-κB in FaDu human pharyngeal cells10 and the Caco-2 human intestinal epithelial layer,12 and attenuate LPS-induced TNF-α gene expression in U937-derived human macrophages.12 On the other hand, L. helveticus MIMLh5 was demonstrated to trigger the secretion of TNF-α and IL-2 once in contact with murine BMDCs.10

It was reported that elderberry fruit extracts may increase the ability of L. acidophilus NCFM to induce interferon (IFN)-β production by BMDCs.43 Inspired by this research, here we investigated the effect of BbE on the stimulatory potential of the MIMLh5 strain on BMDCs. However, in contrast to the results presented by Frøkiær et al.,43 we found that BbE slightly but significantly reduced the ability of MIMLh5 to induce the expression of IFN-β by BMDCs, which may explain the reduced production of IL-12, as well as TNF-α. The different results can be plausibly due to extracts having different content, which must be expected as other plant materials were used here.43

5. Conclusions

In this study, we demonstrated that different food-grade strategies may be potentially adopted to prevent infections and inflammation of the pharyngeal mucosa. The proposed strategies can be employed in a food formulation (e.g., a candy lozenge), whose functional ingredients may potentially prevent GAS colonization (through the effect of guar gum and MIMLh5 probiotic cells) and modulate local immune defenses (through the activity of MIMLh5 probiotic cells and blueberry extract). Such food preparations may be particularly effective for the protection of the pharyngeal mucosa and easily acceptable by school-age children. Nonetheless, this was a proof-of-concept study, and further investigations, both in vitro and in vivo, are needed to define, primarily, the most suitable concentrations of polysaccharide, probiotic and berry extract to use in combination.

Conflicts of interest

There are no conflicts of interest to declare.


Part of the study was financially supported by the “piano di sviluppo UNIMI 2014/2017 (Linea B)” (MAGIC-MAMPs project) of the University of Milan.


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