Anti-inflammatory and macrophage polarization effects of Cranberry Proanthocyanidins (PACs) for periodontal and peri- implant disease therapy

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Periodontal Research published by John Wiley & Sons Ltd Shahram Ghanaati and Frank Schwarz contributed equally. 1Department of Oral Surgery and Implantology, Carolinum, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany 2Post-Graduate Program in Implant Dentistry (PPGO), Federal University of Santa Catarina (UFSC), Florianópolis, Brazil 3FORM-Lab, Frankfurt Oral Regenerative Medicine, Department for Oral, CranioMaxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt, Germany 4Department of Oral Surgery, Universitätsklinikum Düsseldorf, Düsseldorf, Germany 5Department for Oral, Cranio-Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany


| INTRODUC TI ON
Periodontitis is an inflammatory disease caused by anaerobic gram-negative pathogens involving the active production of pro-inflammatory cytokines which modulate periodontal tissue destruction and trigger bone resorption. [1][2][3] Comparably, peri-implantitis is a pathologic condition associated with biofilm formation and is characterized by "inflammation in the peri-implant mucosa and a progressive loss of supportive bone." [4][5][6][7][8] Strong evidence suggests that periodontitis is a risk factor for peri-implant disease. 5,9 Periodontitis and peri-implantitis have similar clinical and radiographic features but evident molecular and histopathological differences. [10][11][12][13] The reported distribution of inflammatory cells in periodontitis and peri-implantitis lesions exhibited that macrophages represent 6% and 11% of the inflammatory cell population, respectively. 12 Even though macrophages are present in moderate proportions at periodontitis and peri-implantitis sites, they play a substantial role in the host's inflammatory response and are considered as the "bridge between inflammation resolution and tissue repair." 14 The dual mechanisms of macrophages concerning turn over and tissue repair might govern homeostasis and disease progression at periodontal and peri-implant tissues 14-16. In this setting, macrophages activated by bacteria sub-products like lipopolysaccharides (LPS) present a M1 phenotype and are associated with the secretion of pro-inflammatory cytokines (ie, IL-1ß, IL-6, IL-8) which trigger a "destructive osteolytic inflammation." Conversely, macrophages activated by alternative ways present a M2 phenotype and are associated with "constructive inflammation" involving the secretion of anti-inflammatory cytokines and growth factors which enhance tissue repair. [17][18][19] Macrophage phenotype and cytokine expression may imply the host's susceptibility to inflammation and response to periodontal and peri-implant therapy. 15,20 Local delivery of host-modulating agents can be a favorable strategy to counteract the pathogenesis and destructive mechanisms of periodontal and peri-implant diseases 2,20-24. Particularly, cranberry (Vaccinium macrocarpon) polyphenolic compounds named proanthocyanidins (PACs) are reported to inhibit oral biofilm adherence, stimulate antibiotic effects, and be potential anti-inflammatory macrophage modulators. [24][25][26][27][28][29][30][31] Accordingly, it is hypothesized that cranberry concentrates may have potential anti-inflammatory effects able to counteract macrophage's "destructive" inflammatory response. The aim of this in vitro study was to evaluate cell viability, anti-inflammatory activity, and macrophage polarization effects of cranberry-derived concentrates.

K E Y W O R D S
anti-inflammatory agents, cranberry, interleukins, macrophage polarization, peri-implantitis, periodontitis, proanthocyanidin were removed by aspiration and adherent cells were cultured in fresh RPMI medium, 10% FCS, for 24 hours at 37°C in 5% CO 2 atmosphere.

| Cell viability assay
Cultured SAOS-2 cells, HGF, and PMA-induced macrophages were exposed to cranberry concentrates at 25, 50, and 100 μg/mL for 2 minutes, 1 hour, and 24 hours and incubated after exposure for 24 hours at 37°C in a humidified 5% CO 2 atmosphere. Cranberry untreated cells at the same time points served as controls. Cell viability for SAOS-2 cells, HGF, and macrophages was measured at days 0 (immediately after treatment), 3, and 7 after the corresponding exposure periods by the use of a luminescence assay according to the manufacturer's instructions (CEllTiter-Glo, Promega) evaluating the live-cell protease activity in an illuminometer (Victor X3; Perkin-Elmer) at a wavelength of 490 nm.

| Treatment of macrophages and LPS stimulation
Previously seeded PMA-induced macrophages were exposed to cranberry concentrations at 25, 50, and 100 μg/mL for 2 minutes, 1 hour, and 24 hours. Cells incubated without cranberry concentrates were used as controls. After exposure to cranberry concentrates, the culture supernatants were replaced with fresh serum-free RPMI medium and macrophages were stimulated with 1 μg/mL of lipopolysaccharide (LPS) (Sigma-Aldrich, Inc) derived from E coli for 24 hours. Macrophages with no LPS stimulation served as an additional control.

| Macrophage cytokine expression
Following 24 hours of LPS of stimulation, the culture supernatants of control and test groups were collected and stored at −20°C.
The supernatants were assayed for pro-and anti-inflammatory cytokine expression. Enzyme-linked immunosorbent assay (DuoSet ® ELISA Development Systems) was used to determine IL-8, IL-1 ß, IL-6, and IL-10 concentrations according to the manufacturer's protocol (R&D Systems). Optical densities of each well were measured using a microplate reader (Infinite200, TECAN) at a wavelength of 450 nm.

| Macrophage immunofluorescence stain
After supernatant removal, wells with adhered LPS-stimulated macrophages were three times (3x) rinsed with PBS and fixed with 4% buffered formalin (Roti-Histofix 4% acid-free pH7, Carl-Roth) for 10 minutes at room temperature. After fixation, wells were rinsed with PBS (3x) and permeabilized with 0.1% Triton X/ PBS. The mentioned rinsing step (3x) was repeated and double im-

| Semi-quantification of M1 and M2 macrophage polarization
Microscopic images were acquired using an inverted microscope (Nikon Eclipse TS100). Images were extracted using fluorophore channels matching to CD68 (total macrophages), CCR7 (M1 polarization), and CD206 (M2 polarization). Representative areas of each group were photographed (10 independent fields per group) (10x objective). The extracted images were analyzed using a software program (ImageJ software, 1.52a). Macrophage polarization semi-quantification was analyzed as percentages of positive doublestained cells for CD68 and CCR7 (M1) or CD68 and CD206 (M2) over the total number of CD68 positive cells.

| Statistical analysis
Statistical analysis was performed using a commercially available software program (SPSS 26, 19.0). All experiments were performed in triplicate samples. Data were analyzed in terms of means and standard deviations (SD). One-way analysis of variance (ANOVA) was used for comparisons between groups. Post hoc test was performed, and differences were considered as statistically significant at P < .05 and P < .01.

| Cell viability
Cell viability of HGF, SAOS-2 cells, and PMA-induced macrophages exposed to cranberry concentrates for 24 hours is presented in Figure 1. No significant effects on HGF viability were detected in response to any of the cranberry tested concentrations. SAOS-2 cell viability was not affected after 24 hours of exposure at day 0 and day 3 of culture; however, it increased at all cranberry exposed groups at day 7, presenting statistical differences (P < .01) for 25 and 50 μg/mL when compared to the control group. Induced macrophages' viability was not affected by cranberry concentrates at day 0; however, it was significantly reduced at days 3 and 7 after exposure to cranberry concentrates at 25 and 50 µg/mL for 24 hours. Cran 50 µg/mL Cran 100 µg/mL pro-inflammatory expression was significantly downregulated (P < .01) when exposed to cranberry concentrates at 50 and 100 µg/mL for 1 and 24 hours (P < .01). Only the cranberry concentrate at 100 µg/mL revealed an antagonist effect, increasing IL-6 expression after 120 seconds of exposure (P < .05). No significant differences were detected for IL-1 ß expression among the control and test groups. Anti-inflammatory IL-10 expression was significantly increased at cranberry 100 µg/mL concentrate when compared to the cranberry untreated control group after 24 hours of exposure (P < .01).

| Effect of cranberry PACs on macrophage polarization
Macrophage M1 and macrophage M2 semi-quantification is pre- blasts previously stimulated with IL-17. 35 Additionally, a similar study exposed a significant downregulation of IL-8 expression in three synovial fibroblasts lines exposed to cranberry NDM at concentrations of 25, 50, 100 and 250 µg/mL. 38 The aforementioned studies correlate with the present results, also pointing to a significant downregulation of IL-8 when macrophages were exposed to cranberry concentrates at 50 and 100 µg/mL for 1 and 24 hours. There is limited evidence evaluating pro-inflammatory IL-1 ß expression after cranberry stimulation. Bodet et al exhibited an antagonist effect of cranberry NDM when exposed to LPS-stimulated macrophages. The cytokine expression increased when macrophages were treated with cranberry NDM at 10 and 25 µg/mL; however, at a concentration of 50 µg/mL, there was a significant reduction of IL-ß secretion. 25 In contrast, the present analysis revealed that cranberry concentrates did not induce any significant effect on IL-1 ß production. IL-6 expression was also evaluated in previous studies. 33,35 A prior study revealed a significant reduction of IL-6 expression when LPS induced macrophages were exposed to cranberry NDM concentrations at 25 and 50 µg/mL for 24 hours. 25 Similarly, Tipton et al revealed a reduction of IL-6 expression by synovial fibroblasts exposed to cranberry NDM at concentrations of 25, 50, 100, and 250 µg/mL. 38 Moreover, Denis et al reported that LPS-stimulated human epithelial colorectal adenocarcinoma cells decreased IL-6 expression when exposed to low, medium, and high molecular mass (250 µg/mL) cranberry compounds. 36 The latter results correlate with the present findings, pointing to a significant downregulation of IL-6 secretion when exposed to cranberry concentrates (A-type PACs) at 50 and 100 µg/ mL for 1 and 24 hours. Even though cranberry-derived concentrates induced a significant downregulation of pro-inflammatory cytokines in the present in vitro study, it is imperative to consider that inflammation on macrophages was induced by a commercially available LPS (E coli) agent which does not resemble the real conditions that exacerbate inflammation at periodontal and peri-implant tissues.
Bearing in mind this limitation, future studies should consider to use LPS derived from biofilm involving periodontitis and peri-implantitis pathogens to mimic a more realistic condition.
Considering the anti-inflammatory cytokine expression, IL-10 was reported to have potential anti-inflammatory properties associated with M2 macrophage activation. 14,15 The present study revealed a significant upregulation of IL-10 expression when macrophages were exposed to the highest cranberry concentrate for 24 hours. These effects need to be further investigated in future studies. In this hallmark, Th1 and Th17 cells have been related to disease progression, upregulating inflammation and osteolytic effects. On the other hand, Th2 cells and Tregs have been described to counteract disease progression and to downregulate inflammation. In this context, the Th transition from Th1 to Th2/Tregs has been associated to M2 polarization induction and the arrest of disease progression. 14,39 Moreover, the host's inflammatory response to bacteria endotoxins as LPS is a critical factor activating "destructive" inflammation at periodontal and peri-implant tissues. Several studies reported that LPS is a potential M1 inductor. [40][41][42] Accordingly, the present study PACs may serve as a potential anti-inflammatory component that can target macrophages enhancing "constructive inflammation" at diseased tooth and implant sites. Nevertheless, future clinical research is indispensable to evaluate the "in vivo" anti-inflammatory effects of cranberry agents.

ACK N OWLED G EM ENTS
The present study was supported by the Osteology Foundation, University, Düsseldorf, Germany, in conducting parts of the analyses.

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest related to this study.