Effects of polyphenolic maqui ( Aristotelia chilensis ) extract on the inhibition of NLRP3 inflammasome and activation of mast cells in a mouse model of Crohn's disease-like colitis

Effects of polyphenolic maqui (Aristotelia chilensis) extract on the inhibition of NLRP3 inflammasome and activation of mast cells in a mouse model of Crohn’s disease-like colitis Tamara Ortiz-Cerda 1,2 *, Federico Argüelles-Arias 3,4 , Laura Macı ´as-Garcı ´a 1 , Victoria Va ´zquez-Roma ´n 1 , Gladys Tapia 5 , Kangzhe Xie 2 , Marı ´a Desire ´e Garcı ´a-Garcı ´a 4 , Manuel Merinero 1,6 , Josefa-Marı ´a Garcı ´a-Montes 3 , Ana Alcudia 6 , Paul K. Witting 2 and Manuel De-Miguel 1 * 1 Departamento de Citologı ´a e Histologı ´a Normal y Patolo ´gica, Facultad de medicina, Universidad de Sevilla, Seville, Spain, 2 Redox Biology Group, The Charles Perkins Centre, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia, 3 Departamento de Medicina, Facultad de Medicina, Universidad de Sevilla, Seville, Spain, 4 Department of Gastroenterology, University Hospital Virgen Macarena, Seville, Spain, 5 Molecular and Clinical Pharmacology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile, 6 Departamento de Quı ´mica Orga ´nica y Farmace ´utica, Universidad de Sevilla, Seville, Spain Introduction: Crohn’s disease (CD) involves activation of mast cells (MC) and NF- кB in parallel with the PPAR-a/NLRP3 inflammasome/IL-1bpathway in the inflamed colon. Whether polyphenols from maqui (Aristotelia chilensis) represent a natural alternative treatment for CD is unclear. Therefore, we used an animal model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD-like colitis to investigate protective effects of maqui extract through monitoring NLRP3 inflammasome and MC activation in colon tissue. Methods: Maqui extract was administered via orogastric route to mice after (post-Treatment group) or prior (pre-Treatment group) to TNBS-induction. Colon pathology was characterized by histoarchitectural imaging, disease activity index (DAI), and assessing NF-кB, p-NF-кB, PPAR-a/NLRP3 expression and IL-1blevels. Results: Compared to mice treated with TNBS alone administration of anthocyanin-rich maqui extract improved the DAI, colon histoarchitecture and reduced both colon wet-weight and transmural inflammation. Induction with TNBS significantly increased colonic NLPR3 inflammasome activation, while cotreatment with maqui extract (either post- or pre-Treatment) significantly downregulated NLRP3, ASC and caspase-1 levels, which manifested as reduced colonic IL-1blevels. Supplemented maqui extract marginally diminished NF-кB activity in epithelial cells but reached statistical significance in immune cells (as judged by decreased NF-кB phosphorylation). PPAR-a Frontiers in Immunology frontiersin.org01 OPEN ACCESS EDITED BY Jeremy P. McAleer, Marshall University, United States REVIEWED BY Dmitriy Smolensky, United States Department of Agriculture (USDA), United States Melinda E. Varney, Marshall University, United States *CORRESPONDENCE Tamara Ortiz-Cerda [email protected] Manuel De-Miguel [email protected] RECEIVED 27 May 2023 ACCEPTED 21 December 2023 PUBLISHED 12 January 2024 CITATION Ortiz-Cerda T, Argüelles-Arias F, Macı ´as-Garcı ´aL,Va ´zquez-Roma ´nV,Tapia G, Xie K, Garcı ´a-Garcı ´a MD, Merinero M, Garcı ´a-Montes J-M, Alcudia A, Witting PK and De-Miguel M (2024) Effects of polyphenolic maqui (Aristotelia chilensis) extract on the inhibition of NLRP3 inflammasome and activation of mast cells in a mouse model of Crohn’s disease-like colitis. Front. Immunol. 14:1229767. doi: 10.3389/fimmu.2023.1229767 COPYRIGHT © 2024 Ortiz-Cerda, Argüelles-Arias, Macı ´as-Garcı ´a, Va ´zquez-Roma ´n, Tapia, Xie, Garcı ´a-Garcı ´a, Merinero, Garcı ´a-Montes, Alcudia, Witting and De-Miguel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 12 January 2024 DOI 10.3389/fimmu.2023.1229767 signaling was largely unaffected by Maqui whereas MC infiltration into the colon mucosa and submucosa decreased and their level of degranulation was suppressed. Conclusion: These outcomes show the post- and pre- Treatment effect of a polyphenolic extract rich in anthocyanins from maqui the acute phase of TNBS- induced CD-like colitis is linked to suppression of the NLRP3 inflammasome and reduced MC responses. These data indicate that maqui extract represents a potential nutraceutical for the treatment of inflammatory bowel disease (IBD). KEYWORDS Crohn’s disease, polyphenols, anthocyanins, maqui, mast cells, NLRP3 inflammasome, interleukin 1b 1 Introduction Inflammatory bowel disease (IBD) represents a cluster of chronic systemic intestinal disturbances that comprise Crohn’s disease (CD), ulcerative colitis (UC), and indeterminate colitis (CI) (1). The clinical course of IBD is characterized by unpredictable periods of cyclic remission and relapse (2). Although the precise pathogenesis of IBD is unclear, it is commonly accepted that an unregulated immune system, dysregulated intestinal microbiota, genetic predisposition, and environmental stimuli are precipitating factors (3). The immune mechanism underlying CD and UC pathogenesis is considered to be mediated by Th1 and Th2, respectively (4). The experimental mouse model using 2,4,6-trinitrobenzene sulfonic acid (TNBS) elicits a colitis-like phenotype with histopathological and morphological changes related to CD. Thus, TNBS administration stimulates a heightened Th1-Th17 response and promotes characteristic histological changes in the colon such as loss of tissue architecture, and transmural immune cell infiltration, which closely resembles important histopathological aspects of human CD (5). Mononuclear hematopoietic and intestinal epithelial cells serve as the first line for colon defense and mediate the innate immune response stimulated by microbes or signaling through pattern recognition receptors (PRR) such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain protein-like receptors (NLRs), or inflammasome proteins such as the NLR family pyrin domain (NLRP1, NLRP3 and NLRC4) and absent in melanoma 2 (AIM2) (6). Thus, inflammasomes are comprised of multiproteic cytoplasmic complexes that assemble via the action of the adaptor protein apoptosis-associated speck-like protein (ASC) and procaspase-1. Activated caspase 1 cleaves pro-interleukin (IL)-1band pro-IL-18 to yield mature pro-inflammatory IL-1band IL-18, respectively (7). Specifically, IL-1bis primarily produced by infiltrating immune cells such as monocytes and dendritic cells as well as mature macrophages in the colon lamina propria, which explains the local and systemic effect of the IBD (8,9). Of the proteins comprising the inflammasome, NLRP3 is the most studied receptor and considered a crucial regulator of intestinal homeostasis that is critically involved in IBD pathogenesis and disease progression (10,11). The presence of lipopolysaccharides (LPS), UV light, and reactive oxygen species (ROS) can activate endogenous transcription factors such as nuclear factor kappa-B (NF-кB), which drive the expression of inflammatory molecules, including the components of NLRP3 (12, 13). Concomitant with transcriptional activation of NF-кB, the transcription factor peroxisome proliferator activated receptorgamma (PPAR-a) is stimulated by microbial factors, tumor necrosis factor-a(TNF-a), LPS, and ATP (14,15). PPAR-ais a master regulator for the transcription of target genes, inhibitor of immune cell activation and protective effects linked to inflammatory processes mediated by NF-кB(16). For example, Palenca I., et al. (2022) demonstrated that the downregulation of the TLR-4/NLRP3 pathway in experimental UC is dependent on PPAR-aactivation (17). Resident colonic mast cells (MC), classified as Tryptase (MC T ) and tryptase and chymase type (MC TC ), can release proteases to damage the mucosal barrier and accumulate in different colonic compartments during IBD pathogenesis (18). For example, increased MC in hypertrophic and fibrotic muscularis propria (19) and MC accumulation in the mucosa,submucosa and muscularis propria is documented in patients with active CD (20). Furthermore, accumulating MC are detected in colons from CD and UC subjects with irritable bowel syndrome (IBS)-like symptoms, and subjects diagnosed with IBS compared to age-, sex-, duration and extent of disease-matched controls (21). Emerging studies have also reported that intestinal MC T and NLRP3 inflammasome activation occur in parallel thereby, implicating a critical role for MC in colon inflammation (22). Conventional treatments for patients with UC or CD have side effects with variable severity and there is no definitive cure. Therefore, the use of nutraceutical or dietary supplements such as probiotics and polyphenols may represent promising alternatives or Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org02 an adjunctive to current therapies. Several studies using murine models of IBD have demonstrated that bioactive polyphenols such as epigallocatechin-3-gallate (EGCG) (23), curcumin (24)or resveratrol (25) act via anti-inflammatory and antioxidant properties, as well as impacting the gut microbiome ultimately maintaining intestinal health and colon-barrier function. Recent studies with maqui fruit (Aristotelia chilensis) have focused on its potential health benefits attributed primarily to anthocyanin polyphenols (26). We have shown previously that maqui fruit extract contains a high polyphenolic content and is rich in anthocyanins, especially delphinidins (27). In vitro studies with epithelial cells and macrophages demonstrated that maqui extract has noncytotoxic effects, and powerful antioxidant capacity which contribute to its anti-inflammatory action (27). Supplemented maqui extract significantly ameliorated the clinical course of the disease and the extent of intestinal inflammation in an experimental animal model of CD. Thus, maqui provided either post- or pre- Treatment down-regulated COX-2 and iNOS protein expression, up-regulated nuclear factor E2-related factor 2 (Nrf-2)/antioxidant heme oxygenase 1 (HO-1) signaling pathway and altered macrophage polarization towards the inflammation resolving M2 phenotype (28). Overall, the aim of this present study is to explore the anti-inflammatory effects of a polyphenolic extract rich in anthocyanins from Aristotelia chilensis through inhibition of NLRP3 inflammasome and MC activation using an animal model of TNBS-induced CD-like colitis. 2 Materials and methods 2.1 Polyphenolic maqui extract and treatment dosage To obtain a large-scale quantity of maqui berry extract, the following extraction procedure was performed using acidified methanol at pH 1 (MeOH/H + 0.1% v/v HCl) with the primary goal to prevent chemical changes and preserve polyphenolic components (27). Thus, all workup procedures were conducted in the dark and controlled temperature (room temperature during the collection of organic phase and 35°C during solvent evaporation). The final product was lyophilized and stored at -20°C. Where required, lyophilized polyphenolic maqui extract was dispersed in water and administered via orogastric tube (total amount 50 mg/kg/ day) given in a single daily dose as per the four treatment groups outlined below. 2.2 Induction of acute Crohn’s disease and experimental design This study was approved by the Ethical Committee of the Faculty of Medicine, University of Seville and the animal handling was conducted in accordance with the Guide for the Care and Use of Laboratory Animals [22]. Experimental colon disease was induced with TNBS (Sigma-Aldrich; administered at 100 mg/Kg body weight) delivered through an intracolonic path as reported previously (28). Male Balb/c mice 12-14 weeks old were randomly assigned in 4 groups (n=6) as follows: 1) Control Group: Mice were undergoing to intra-rectally intervention using ethanol (EtOH 50%) neither TNBS nor maqui extract. 2) Crohn’s disease-like Colitis Group (CD group): 100 mg/kg of TNBS plus EtOH 50% without maqui extract. 3) post-Treatment Group: Maqui extract was administrated for 4 days after TNBS induction. 4) pre-Treatment Group: Maqui extract was administrated for 7 days prior TNBS and 4 days after TNBS induction. 2.3 Disease activity index and colon macroscopic evaluation The disease activity index (DAI) score was obtained following the criteria published previously by Gommeaux et al. (29). The scoring system was used for monitoring the experimental mice from day 0 until the end of experiment to quantify disease progression. Thus, the extent of diarrhea, rectal bleeding, and weight loss were monitored daily and graded from 0 to 3 consistently by the same researcher to yield a total score that constitutes the DAI. At the end of the experiment, the average total DAI score was determined to capture the gross progression of disease. After 5 days, mice were anesthetized, the abdominal cavity was opened and the presence of adhesion and structural variations of large intestine such as ulcerations, oedema and changes in the stool consistency were documented. Subsequently, the isolated colon was rinsed with PBS and cleaned of stool to ascertain wet weight. Next, images of colonic morphology were captured using a Canon EOS 350 zoom camera (Canon Inc. Tokyo, Japan). Thereafter, the large intestine was cut in two halves longitudinally. One half was assigned to histopathological analyses and immediately fixed with 4% v/v buffered paraformaldehyde while the remaining colon was frozen in liquid nitrogen and assigned for biochemical analyses. 2.4 Histopathological study For histopathological examination, the large intestine was dissected and rolled from the distal to the proximal end to form a “swiss roll”. The longitudinal fractions of the colon of each animal were fixed in 4% v/v buffered paraformaldehyde and embedded in paraffin. Thereafter, sections of tissue (4 mm thickness) were cut using a rotary microtome (Microm HM 310, Thermo Scientific), mounted on glass slides and dried (2 h, 60°C) before staining with different techniques. All histology slides were analyzed in a blinded fashion by two pathologists using an Olympus microscope (Vanox AHBT3, Tokyo, Japan) equipped with a Nikon DS-Fi3 camera. Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org03 2.4.1 Hematoxylin & eosin staining Where required, colon sections were deparaffinized, hydrated and stained with hematoxylin–eosin according to standard protocols. All stained sections were imaged, and characteristic histopathological changes were examined including inflammatory infiltrate, loss of histoarchitecture, presence of ulcerations, presence of crypt abscesses and cellular necrosis. All sections were assessed at image magnifications of 40X and 200X to conduct histology for the whole organ. 2.4.2 Toluidine blue staining and quantification of mast cells To evaluate the number and location of MC, colon tissue was stained with toluidine blue stain. Briefly, samples were immersed in a toluidine blue solution at 0.5% w/v toluidine blue (Sigma-Aldrich- Germany) in distilled water acidified with glacial acetic acid at 1% v/v for 5 min. Subsequently, sections were rapidly dehydrated with ethanol (90 and 100% each x 1 min). After two further consecutive washes in xylene (2x and 5x min), stained sections were coverslipped using DPX mountant and visualized using an Olympus photomicroscope allowing for complete imaging from the distal to the proximal colon end. Cells detected were identified as either tryptase MC (MC T ) or tryptase and chymase MC (MC TC )basedon their localization in mucosa or submucosa connective tissue, respectively. Therefore, frequency and morphology characteristics including size and degranulation were evaluated. The proportion of MC subtype in each compartment was finally expressed as a percent (%) of total cell population in the relevant compartment. 2.5 Immunohistochemistry studies Immunostaining for NF-kB, p-NF-kB and PPAR-awas performed in colon sections after dewaxing and rehydration followed by heat-induced antigen retrieval. During the antigen retrieval process, EDTA buffer pH 8.0 was used for NF-кB and phospho-NF-кB (pNF-кB) whereas citrate buffer pH 6.0 was used for PPAR-a. Endogenous peroxidase activity and non-specific binding was blocked with hydrogen peroxide and normal horse serum, respectively. Mouse IgG blocking with ReadyProbes™ Mouse on Mouse IgG Blocking Solution was conducted for NF- кB. Incubation with primary anti-NF-кB (Thermo Fisher Scientific Cat# PA1-30408, RRID : AB_1958961), anti-p-NF-кB (Santa Cruz Biotechnology Cat# sc-136548, RRID : AB_10610391) and anti- PPAR-a(Affinity Biosciences Cat# AF5301, RRID : AB_2837786) were performed overnight at 4°C. Secondary antibodies were obtained from the ImmPACT DAB kit (Vector, USA) following manufacturer’s recommendations. Mayer’s Hematoxylin was used for nuclear contrast (Modified solution according to Lillie, ScyTek Laboratories, Utah, U.S.A). Analysis of positive nuclei was performed under light microscope in a blind fashion in 10 adjacent (400X magnification) per slide. The intact area and inflammatory regions were select and saved for immunostaining. Scoring of immunopositive + stained sections was performed as described previously (30,31). To evaluate NF-кB and p-NF-кB expression in epithelial cells, the total percentage of positive cells (200x magnification) were recorded within representative areas for each isolated colon and averaged across all colons in the same treatment group. For PPAR−aexpression, scoring was according to the proportion of epithelial cells that stained immunopositive + for PPAR−aexpression determined as a percentage, yielding corresponding pathological scores of 1 for <25% of cells being stained, 2 for 25−50% and 3 for >50% cells immune + for PPAR−a. The cytoplasmic expression of PPAR-a, NF-кBandp-NF-кB in macrophage cells was performed by selecting hotspots and manually counting cells across 10 fields (@400x magnification) per slide. Overall, the extent of pathology was scored independently by 2 blinded experts and the compiled data averaged. 2.6 Isolation of cytoplasmic proteins and western blot assay Frozen colonic tissues were randomly selected (4 per group), weighed and homogenized in ice-cold buffer (containing: 50 mM Tris-HCl, pH 7.5, 8 mM MgCl 2 , 5 mM ethylene glycol-bis (2- aminoethylether)-N,N,N´,N´-tetraacetic acid, 0.5 mM EDTA, 0.01 mg/mL leupeptin, 0.01 mg/mL pepstatin, 0.01 mg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 250 mM NaCl). The homogenates were centrifuged at 12,000 × g, 4°C for 15 min, and the clarified supernatants were collected and stored at −80°C. Protein concentration of the colon homogenates was determined by Bradford’s method (32). Aliquots of supernatants containing equal amounts of protein (normalized to 30 mg) were separated on 10% acrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis then transferred onto a nitrocellulose membrane, which was then stained with Ponceau red to confirm equal loading of total proteins. Next, the membranes were blocked with 5% BSA in Nonidet™at 0.5% and PBS. Then, the membranes were incubated with specific primary antibodies: rabbit anti-NLRP3 (final dilution 1:1000 v/v; Cell Signaling Technology Cat# 15101, RRID : AB_2722591), rabbit anti-ASC (final dilution 1:1000 v/v; Cell Signaling Technology Cat# 70891, RRID : AB_2799791) and rabbit anti-caspase 1 (final dilution 1:1000 v/v; Abcam Cat# ab1872, RRID : AB_302644) overnight at 4°C. After rinsing, the membranes were incubated with the horseradish peroxidase-linked (HRP) secondary antibody anti-rabbit (1:1000 v/v; Cell Signaling Technology Cat# 7074, RRID : AB_2099233) Cayman Chemical, Ann Arbor, MI, USA) or anti-mouse (1:1000 v/v; Agilent Cat# P0447, RRID : AB_2617137) containing blocking solution for 1 h at room temperature. To prove equal loading, the identical blots were also analyzed for b-actin expression using an anti-b-actin antibody (1:1000 v/v; Sigma Aldrich, St. Louis, MO, USA). All immunodetection was performed using an enhanced chemiluminescence light-detecting kit (SuperSignal West Pico Chemiluminescent Substrate, Pierce, IL, USA). Immunosignals were captured using Amersham imager 600 (Healthcare Life Sciences, Buckinghamshire, UK) and densitometric data were studied following normalization to the housekeeping protein such as b-actin used as an internal control. The signals were analyzed and Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org04 quantified by an Image Processing and Analysis in Java (Image J, Softonic ®, National Institute of Mental Health, Bethesda, MD, USA) and expressed relative to the DSS-exposed control group (arbitrarily assigned as 100%). 2.7 Determination of IL-1 bproduction Production of colonic IL-1bwas determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Murine IL-1b, PeproTech, Cranbury, USA) according to the manufacturer’s protocol. Absorbance determined at 415 nm with a microplate reader (Sinergy HT, Biotek®, Bad Friedrichshall, Germany). Levels of this cytokine were determined in duplicate and expressed as picograms per milligram of wet weight tissue (pg/mg tissue). 2.8 Statistical analysis All data were represented as means ± SEM and analyzed using GraphPad Prism 8.0 program (GraphPad Software, San Diego, Canada). In all cases, heterogeneity was tested by using Levene’s test and Shapiro-Wilk test for normalcy. For between group comparison of means (n=3 technical repeats), parametric data were analyzed by One-way analysis of variance (ANOVA) whereas a Kruskal-Wallis test was used for any non-parametric data sets, followed by Bonferroni’spost hoc test. p-values< 0.05, <0.01 or <0.001 were considered statistically significant. 3 Results 3.1 Treatment with maqui extract rich in Delphinidin improve clinical outcomes and macroscopic parameters in acute TNBS- induced Crohn’s disease-like colitis Data in Figure 1A shows the previously published proportion of polyphenolic compounds from the maqui extract (27). Phenolic compounds separated by UHPLCMS/MS identified high levels of Delphinidin (70.4%) and Cyanidin (28.8%), mainly comprising Delphinidin-3-O-glucoside 34.3% and Cyanidin-3-O-glucoside 8.3%, respectively, along with other anthocyanins such as Malvidin-3-O-glucoside (0.8%), Pelargonidin-3-O-glucoside (0.1%) and Peonidin-3-O-glucoside (0.1%). To test the effect of this maqui extract we carried out an intervention using an experimental model of CD-like colitis using TNBS to trigger acolonicinflammatory response that closely resembles immunological (33) and histopathological aspects of human CD (28). The daily clinical index increased significantly in mice exposed at TNBS at day 5. Whereas, this index was attenuated significantly in the post- and pre-Treatment groups, that typically showed diminished rectal bleeding and improved stool consistency (Figure 1B). As anticipated, the mice from the CD group showed a notable decline in percent body weight post TNBS administration. As previously reported (28), mice assigned to the post-and pre- Treatment groups both recorded a lower extent of weight loss at day 2 of treatment compared to mice treated with TNBS alone. Notably, mice treated with maqui extract prior to TNBS treatment showed a recovery in body weight from the point of disease induction until sacrifice (refer to reference (28) and Figure 1C), At the intervention end point (day 5), mice from the experimental CD group showed a significant percent body weight loss (decreasing ~19% vs the control group). By contrast, mice treated with maqui extract either by post- or pre-interventions consistently presented with less weight loss (2.5% and 0.1%, vs control, respectively) (Figure 1D). At day 5, a significant increase in body weight-corrected colon weight was determined (2.9%) in the experimental CD-like colitis group in the absence of maqui extract. By contrast, the increase in colon weight from mice co-treated with TNBS and maqui extract was markedly lower (1.1% and 1.3% for post- and pre-Treatment groups vs control, respectively) consistent with the natural product inhibiting tissue oedema and inflammation (Figure 1E). Macroscopically, extensive hyperemia, marked bowel wall thickening and adhesions between the colon and adjacent tissues was observed in colons isolated from the experimental CD-like colitis group (Figure 1Fa, b, c and Supplementary data S1). All these pathological parameters were diminished by supplementation with the extract (compare images d, e, f from Figures 1F). 3.2 Maqui extract alleviates histology damage in acute experimental animal model of Crohn’s disease-like colitis Severe and extensive transmural infiltration of neutrophils and eosinophils was detected in the colon mucosa and submucosa from mice with experimental CD-like colitis relative to the control group and in the absence of maqui (compare Figures 2A,Band Supplementary S2). This extensive infiltration was accompanied by a marked distortion of colon architecture, with evidence of focal necrosis and goblet cells depletion. By contrast, colons from mice co-treated with TNBS and maqui extract were consistently protected from inflammation as evidenced by a decrease in inflammatory cell infiltration and recovery of intestinal architecture (Figure 2C). Atrophic mucosa and loss of cytoplasm were still observed in mice from the post-Treatment group albeit at lower extents of focal glandular atrophy and mucosal crypts loss. Therefore, signs of chronic inflammation remained in these tissues (Figure 2D). 3.3 Maqui extract reduced the activation of inflammasome NLRP3 and decrease the colon IL-1blevel in the colons of mice with acute TNBS-induced Crohn’s diseaselike colitis An aberrant activation of NLRP3 inflammasome and an overregulation of cytokines IL-1band IL-18 have been reported Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org05 to be essential in the early phase of the inflammatory cascade in IBD (34). Data obtained here showed that TNBS stimulation in the absence of maqui markedly increased colon NLRP3 levels, while cotreatment with maqui extract, both as post- and pre-Treatment groups significantly downregulated NLRP3 levels in mice (Figure 3A). As indicated in Figures 3B,C, protein expression of ASC and Caspase 1 in the colon tissue from the experimental CD group trended to increase, although this was not significantly different to the control group. Overall, treatment with 50 mg/kg/d of maqui extract suppressed the expression of these proteins. Similarly, IL-1blevels increased significantly in mice with experimental CD-like colitis. In contrast, colon IL-1bwas significantly reduced in the post- and pre-Treatment groups (Figure 3D). These data showed that maqui extract actively B CD EF A FIGURE 1 Maqui extract rich in Delphinidin prevent weight loss, improve clinical outcomes and macroscopic parameters in experimental animal model of Crohn’s disease-like colitis induced by TNBS. (A) Distribution of polyphenolic compounds in maqui extract as determined by (UHPLC-MS/MS) Orbitrap. The data are expressed as a proportion of polyphenol content. The effect of orogastric maqui extract on (B) DAI score recorded throughout the entire experiment, (C) Percent body weight loss in mice over the experiment and until sacrifice (28), (D) Percent body weight at day 5 (culling day), (E) Colon weight as percent of body weight on day 5 (F) Representative macroscopic image from the entire intact colon isolated from CD group (a,b,c), Control group (d), post- and pre-Treatment groups (f) and Supplementary Figure S1. Values represent mean ± SEM (n=6). p-value < 0.05, p-value < 0.001 and p-value < 0.0001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test are shown between different experimental groups. Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org06 inhibited components of multiprotein complex associated with NLPR3 inflammasome activation, and this was linked to decreased IL-1blevels in the same colons, suggesting that in addition to acting via antioxidant mechanisms, this natural product also provides an anti-inflammatory effect. 3.4 Maqui extract treatment weakly modulates the transcriptional factor NF-кB through diminishing NF-кB phosphorylation in colon epithelial cells The transcription factor NF-кB plays a key role in mediating IBD pathologies. Once phosphorylated, NF-кB translocates from the cytoplasm to the nucleus, which subsequently stimulates NRLP3 and initiates production and secretion of proinflammatory cytokines IL-1band IL-18, which contribute to the aberrant immune response observed in IBD (35). Data shown in Figures 4A,Bindicated that TNBS induction resulted in more intense immune + -NF-кB staining and yielded an increased proportion of NF-кB positive epithelial cells compared to the control group (reaching 1.93-fold increase in mean levels), although this was not statistically significant (Figure 4E). After treatment with maqui extract, both post- and pre-Treatment groups, showed a weak trend to down-regulated immune + -NF-кB staining and intensity of the cytosolic NF-кB staining in epithelial cells (Figures 4C,D), which was not statistically significant (Figure 4E). In terms of p-NF-кB expression in the epithelial cells, there was no positive staining of p-NF-кB observed in the colons from the control group (Figure 4F). By contrast, an intense nuclear staining of p-NF-кB and significantly higher proportion of epithelial cell p-NF-кB positive expression was detected in colons from the experimental CD group (Figures 4G,J). Decreased p-NF- кB positive expression and intensity was observed in the groups that received maqui extract treatment, although this trend was not significant (compare Figures 4H-J). 3.5 Modulation of maqui extract on transcriptional factor NF-кB through the prevention of NF-кB phosphorylation in autoimmune cells The cellular expression of NF-кB and p-NF-кB in macrophages was also examined. Similar to the trend observed in the epithelial cells, TNBS-induced experimental CD-like colitis resulted in a B CD A FIGURE 2 Treatment with maqui extract improve histopathological evaluation of colonic tissue from animal model of Crohn’s disease-like colitis after Hematoxylin and Eosin staining (H&E). A representative image from entire “Swiss rolled”colon evaluation from the distal to proximal end at 40x magnification (objective 4) (n= 6 mice/group, see Supplementary Figure S2). (A) Control Group: Normal histology colon tissue. Physiology immune cells in lamina propria (black arrow in 10x). (B) CD Group: Presence of necrosis (black arrow in 40x). and goblet cells depletion (dashed arrow). Transmural infiltrate inflammatory (black arrow in 20x). (C) post-Treatment group: Preservation of the colonic mucosa structure (black arrow in 40x). Normal cells infiltration (black arrow in 20x). (D) pre-Treatment group: Lesser extent of atrophy (black arrow in 4x) and mucosal crypts loss (black arrow in 20x). Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org07 greater cytoplasmic intensity and number of NF-кB positive cells when compared to the control group, although again this was not significant (Figures 5A,B,E). The administration of maqui extract caused a weaker cytoplasmic staining intensity of NF-кB and a marginal reduction in the number of NF-кB positive cells in the post-Treatment group (Figure 5C). In the pre-Treatment group, maqui extract treatment did not alter the staining intensity of NF- кB nor the number of NF-кB positive macrophages (Figure 5D). None of these changes were statistically significant (Figure 5E). Data shown in Figures 5F,Gindicate that the nuclear staining of p-NF- кB was scarce and almost no p-NF-кB positive macrophages were observed in the control mice, whereas the administration of TNBS led to strong nuclear staining of p-NF-кBandsignificantly increased numbers of p-NF-кB positive macrophages in the experimental CD-like disease group (Figure 5J). Under these inflammatory conditions, treatment with maqui extract alleviated the TNBS-mediated increase in p-NF-кB levels, as judged by the weaker nuclear p-NF-кB staining intensity and number of p-NF-кB positive macrophages cells, which reached statistical significance in the post-Treatment group (Figures 5H,I). However, this diminution of p-NF-кB positive cells remained above levels determined in control mice in the absence of TNBS (Figure 5J). Together, these results suggest that Maqui extract modulates the proinflammatory NF-кB transcription activation by inhibiting the extent of phosphorylation during the inflammation induced by TNBS in mice, with a more prominent effect observed in the post- Treatment group. 3.6 Potential anti-inflammatory effect of maqui extract on PPAR-aexpression in epithelial and macrophage cells in colon tissue from experimental model of Crohn’s disease-like colitis. It has been shown that PPAR-aexerts its anti-inflammatory action by negatively influencing the transcriptional activity of NF- кB, and in the absence of PPAR-agene, IBD pathologies were exacerbated (16). Next, we explored whether maqui extract modulated PPAR-aexpression in colon tissue. In the epithelial cells, TNBS induction resulted in a weaker PPAR-aexpression score in the experimental CD-like disease group when compared to the control group (Figures 6A,B). Notably, this difference did not reach statistical significance (Figure 6E). The administration of maqui extract resulted in similar PPAR-astaining intensity and PPAR-aexpression score for both post- and pre-Treatment groups compared to the corresponding control (Figures 6C-E). In terms of specific macrophage PPAR-alevel, a stronger staining intensity and a trend towards elevated PPAR-apositive cells were observed in the CD mice when compared to the controls 118 kDa 42 kDa NLRP3 β-acn 0 50 100 150 cirtemotisneDsisylanaNLRP3 (%) ** A Control Group CD Group post-Treatment Group pre-Treatment Group 0 50 100 150 Densitometric aanalysis ASC (%) 25 kDa 42 kDa ASC β-acn B Control Group CD Group post-Treatment Group pre-Treatment Group 0 50 100 150 cirtemotisneDsisylanaesapsaC-1 (%) 20 kDa 42 kDa Caspase-1 β-acn C 0 10000 20000 30000 40000 50000 IL-1β producon (pg/mg ssue) Control Group CD Group post-Treatment Group pre-Treatment Group Control Group CD Group post-Treatment Group pre-Treatment Group D * ** * * * ** FIGURE 3 Anti-inflammatory effect of the maqui extract (50 mg/kg/d) on inhibition of multiprotein complex inflammasome NLPR3 and pro-inflammatory cytokine IL-1bfrom colons of Crohn’s disease-like colitis (n= 6 mice/group). Representative Western blot images and densitometric analysis of each group: (A) NLR Family pyrin domain 3 (NLRP3), (B) Adaptor protein apoptosis-associated speck-like protein (ASC) and, (C) Caspase 1. b‐actin was used as an equal loading control for normalization. (D) ELISA quantification of IL-1bproduction. Values represent mean ± SEM; *Difference at the p < 0.05 level, **Difference at the p < 0.001 level; one-way ANOVA followed by Bonferroni’s Multiple Comparison test. Ortiz-Cerda et al. 10.3389/fimmu.2023.1229767 Frontiers in Immunology frontiersin.org08 however, this difference was not significant (Figures 6F,G,J). Similarly, an increase in PPAR-astaining intensity and PPAR-a positive macrophages were also observed in mice that received maqui post-induction of the disease (Figures 6H,J), whereas colons from mice receiving maqui extract as a pre-Treatment yielded a strong PPAR-astaining and significantly higher PPAR-apositive macrophages when compared to the controls (Figures 6I,J). These data show that during acute inflammatory processes there is upregulated expression of anti-inflammatory transcription factor in the mouse colon, likely acting as a compensatory mechanism. Also, these outcomes supported the idea that the administration of maqui extract –especially prior to TNBS induction –can potentiate macrophage expression of PPAR-atranscription factor compared to normal physiological conditions and, elicit a weak trend to increase PPAR-aexpression in CD-like disease. This may suggest an anti-inflammatory role through regulating PPAR-aactivation. 3.7 Maqui extract exert suppressed mast cells activation in the colons of mice with TNBS-induced Crohn’s disease-like colitis MC are relevant in the intestinal inflammation, however their precise role and mechanism of action during IBD pathogenesis is still unclear. To explore whether therapeutic effect of maqui extract extends to regulating MC populations in the colon, we conducted toluidine blue staining due its metachromasia properties. An increase in MC number was identified in the colons from the experimental FIGURE 4 Polyphenolic maqui extract shows preventing trend of decreasing NF-кB phosphorylation in colon epithelial cells from experimental animal model of Crohn’s disease-like colitis. Immuno‐histochemistry for NF-кB in colon from all groups (n= 6 mice/group): Immune + -NF-кB staining intensity in cytoplasm from crypts cells (black arrow) in (A) control group, (B) CD group, (C) post-Treatment group and (D) pre-Treatment group (D). Dashed arrow shows positive staining in colon epithelium in the pre-Treatment group (image D). Immuno‐histochemistry for p-NF-кB in colon from all groups: (F) Absence of staining of p-NF-кB in colons from control group (black arrow shows epithelial cell nucleus without positive staining for p- NF-кB), (G) Immune staining of p-NF-кB in crypts cells (black arrow) and in the epithelium (dashed arrow) in CD group, (H) Immune p-NF-кB staining intensity in cytoplasm from colon epithelium (black arrow) in the post-Treatment group (I) Immune p-NF-кB staining intensity in cytoplasm from crypts cells (black arrow) in the pre-Treatment group. 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