Polyphenol-rich diet mediates interplay between macrophage-neutrophil and gut microbiota to alleviate intestinal inflammation

Inflammatory bowel disease (IBD) is characterized by a dysregulated mucosal immune response to the gut microbiota, leading to inflammation, tissue damage, and microbial dysbiosis. Macrophages play a central role in IBD pathogenesis, exhibiting plasticity in differentiation and regulating inflammation. Dysregulated pro-inflammatory macrophage activation in response to gut microbiota alterations contributes to IBD. A shift towards regulatory immune phenotypes in macrophages, including enhanced M2 polarization, can alleviate colitis. The metabolic profile significantly influences macrophage phenotype and function. Precise regulation of macrophage polarization and immunometabolism is crucial for clearing microorganisms, eliminating damaged cells, and driving epithelial cell renewal. The gut microbiota is critical for maintaining gastrointestinal tract integrity, mucosal homeostasis, and host metabolism. Changes in gut microbial composition and metabolites are associated with IBD. Intestinal microbiota is necessary for colitis initiation and development in DSS-treated or IL-10-deficient mice. Restoring gut homeostasis by normalizing the gut microbiota is a therapeutic target for IBD. Fecal microbiota transplantation (FMT) from healthy donors can alleviate the inflammatory response in murine colitis models. Previous research demonstrated that FMT from green tea polyphenol-dosed mice improved intestinal epithelial homeostasis and ameliorated colitis. Phenolic acids, a large group of compounds, improve gut function by reducing intestinal inflammation and altering the gut microbiota. Polyphenols and the microbiota interact to influence intestinal inflammation by regulating macrophage activation and gut microbial metabolites. Chlorogenic, ferulic, caffeic, and ellagic acids critically influence gut health. However, the involvement of the gut microbiota and macrophages in phenolic acid-mediated alleviation of gut inflammation remains poorly understood. This study aimed to investigate the interplay between phenolic acids, gut microbiota, and macrophages in a controlled manner using antibiotic-treated or macrophage-deleted mice.

Extensive research highlights the crucial role of the gut microbiota in IBD pathogenesis and the potential therapeutic benefits of manipulating its composition. Studies have shown a strong correlation between dysbiosis and IBD, with alterations in the abundance of specific bacterial taxa linked to disease severity. FMT has emerged as a promising therapeutic strategy, with several studies demonstrating its efficacy in alleviating symptoms in IBD patients. The precise mechanisms by which FMT exerts its beneficial effects are still under investigation, but it is believed to involve the restoration of a healthy microbial balance and the production of beneficial metabolites. In addition, the role of macrophages in IBD has been extensively studied, with a particular focus on their polarization states and their contributions to both inflammation and resolution. The plasticity of macrophages allows them to adapt to their microenvironment and adopt distinct phenotypes, such as the pro-inflammatory M1 and anti-inflammatory M2 macrophages. Dietary polyphenols, including phenolic acids, have garnered increasing attention for their potential to modulate the gut microbiota and influence immune responses. Studies have shown that certain polyphenols can selectively modulate the growth of specific bacterial species, leading to a shift towards a more balanced and protective microbiota. Furthermore, polyphenols have been shown to have direct effects on immune cells, including macrophages, influencing their polarization and cytokine production.

This study used male C57BL/6 mice housed in specific pathogen-free (SPF) conditions. Acute experimental colitis was induced by administering 3% dextran sulfate sodium (DSS) in drinking water for 5 days, followed by regular water for 3 days. Colitis severity was assessed by monitoring body weight, histological scores, disease activity index (DAI), and colon length. Intestinal microbiota depletion was achieved by administering a four-antibiotic cocktail in drinking water for 2 weeks. Fecal microbiota transplantation (FMT) was performed by orally inoculating mice with fecal microbial suspensions from healthy donors. Macrophage depletion was accomplished by injecting clodronate-loaded liposomes intraperitoneally. Neutrophil depletion was achieved using anti-Ly6G antibodies. Mice were orally administered four phenolic acids (chlorogenic, ferulic, caffeic, and ellagic acids) for 8 days, with DSS treatment starting on day 3. Bone marrow-derived macrophages (BMDMs) were isolated and differentiated for transplantation into macrophage-depleted mice. IL-22 neutralization was performed using an anti-IL22 antibody. Histology, flow cytometry, RT-PCR, ELISA, glycolysis assays, and caspase-1/NLRP3 activation assays were used to evaluate colitis severity, macrophage polarization, neutrophil activity, cytokine production, and metabolic changes. Statistical analyses were conducted using two-tailed Student's t-tests or one-way ANOVA with Tukey's post-hoc test.

The study revealed that the absence of intestinal microbiota and macrophages exacerbated epithelial injury in DSS colitis. Chlorogenic acid (CGA) significantly mitigated colitis severity by reducing M1 macrophage polarization. This effect was mediated by the suppression of pyruvate kinase M2 (Pkm2)-dependent glycolysis and the inhibition of NOD-like receptor protein 3 (NLRP3) activation. The protective effects of CGA were entirely dependent on the presence of macrophages, as demonstrated by the lack of benefit in macrophage-depleted mice. Ferulic acid (FA) also alleviated colitis, but its effect was neutrophil-dependent, primarily through the reduction of neutrophil extracellular traps (NETs) formation. Depletion of neutrophils exacerbated colitis symptoms, and FA failed to mitigate inflammation in neutrophil-deficient mice. Both caffeic acid (CA) and ellagic acid (EA) exerted their anti-inflammatory effects in a gut microbiota-dependent manner. In microbiota-depleted mice, CA and EA supplementation did not alleviate colitis. FMT from CA or EA-treated mice successfully transferred the protective effect, suggesting that the modulation of gut microbiota by these polyphenols plays a crucial role in colitis prevention. Interestingly, the metabolites produced by gut microbiota following EA treatment, particularly urolithin A (UroA), displayed potent anti-inflammatory properties. UroA significantly alleviated colitis symptoms and improved gut barrier function in an IL-22-dependent manner. Neutralization of IL-22 abolished the protective effects of UroA, highlighting the critical role of this cytokine in mediating the anti-inflammatory effects of this microbial metabolite.

This study provides compelling evidence for the complex interplay between dietary polyphenols, gut microbiota, and immune cells in the pathogenesis and treatment of IBD. The findings highlight the distinct mechanisms by which different phenolic acids exert their beneficial effects. CGA's action on macrophage metabolism, FA's effect on neutrophil function, and EA/CA's dependence on gut microbiota-mediated modulation, reveal the multifaceted nature of polyphenol-mediated protection against intestinal inflammation. The identification of UroA as a key metabolite responsible for the protective effects of EA underscores the importance of considering the gut microbiome's metabolic activity when assessing the health benefits of polyphenols. The study's findings advance our understanding of the mechanisms underlying the therapeutic potential of polyphenols in IBD and suggest potential avenues for the development of novel therapeutic interventions. The intricate crosstalk between immune cells and the microbiota emphasizes the importance of a holistic approach to IBD treatment.

This study demonstrates that polyphenol-rich diets alleviate intestinal inflammation through diverse mechanisms involving macrophage-neutrophil interactions and gut microbiota modulation. Chlorogenic acid targets macrophage metabolism, ferulic acid inhibits NET formation, while caffeic and ellagic acids depend on gut microbiota alterations for their effects. Urolithin A, a metabolite of ellagic acid, plays a critical role in gut barrier protection via IL-22. These findings highlight the complex interplay between diet, gut microbiota, and the immune system in intestinal health and offer potential strategies for IBD management. Future research should focus on validating these findings in human studies and exploring the specific molecular targets and signaling pathways involved in the observed effects.

This study utilized a murine model of colitis induced by DSS, which may not perfectly replicate the complexity of human IBD. The dose and administration route of phenolic acids were chosen based on previous studies but may not fully reflect human dietary intake. The study focused on a limited number of phenolic acids and did not investigate the potential synergistic or antagonistic interactions among different polyphenols. Further research is needed to determine the clinical relevance of the findings and to investigate the long-term effects of polyphenol consumption on intestinal health.