Pomegranate peel, as a traditional Chinese medicine, has shown excellent anti-oxidant and anti-inflammatory effects, due to the large number of polyphenols in its components. Punicalagin (PUN) is the most abundant polyphenol component in pomegranate peel, and its anti-oxidant capacity was shown to be more than 50% of the whole pomegranate peel extract (Xu et al., 2021). Many studies have shown that PUN has anti-oxidant and anti-inflammatory effects (Venusova et al., 2021). The anti-inflammatory effect of PUN was manifested as inhibiting the release of pro-inflammatory factors such as nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in macrophages, promoting the polarization of macrophages to M2c type, and enhancing the expression of anti-inflammatory cytokines such as IL-10 and macrophage colony-stimulating factor (M-CSF) (Xu et al., 2014, 2017; Yan et al., 2021). Other studies have shown that PUN also has a variety of other activities, including anti-apoptosis, anti-proliferation, anti-bacterial, anti-viral and anti-tumor activities (Venusova et al., 2021). However, the effect of PUN on macrophage phagocytosis has not been reported.

As phagocytes, macrophages face a diverse array of particles, ranging from pathogens to apoptotic cells, which require a variety of different receptors to recognize, engage, and initiate the phagocytosis of these different targets (Ayaz and Ocakoglu, 2020; Liu et al., 2023; Pidwill et al., 2021). Phagocytosis requires actin-dependent remodeling of the cell membrane to facilitate phagocytic receptor movement, pathogen detection, and phagocytosis (Grinstein, 2014; Hallett, 2023; Zhou et al., 2022). Phagocytic receptors are classified into opsonic and non-opsonic receptors. Opsonized receptors involved in the phagocytosis of opsonin-coated particles, mainly include Fc receptors (FcRs) and complement receptors, which bind to ligands on the surface of pathogens (Xin et al., 2019). However, non-opsonized receptors such as C-type lectin receptor, CD14, CD36, mannose receptor, macrophage receptor with collagen structure (Marco) and scavenger receptor A directly recognize the molecular patterns present on the phagocytosed particles and are involved in the internalization of non-opsonized particles (Goodridge et al., 2012). Different types of phagocytic receptors may contribute to different mechanisms of phagocytosis.

Macrophages in response to the challenge of a large number of pathogens, express C-type lectin receptors (CLRs), which recognize pathogens and participate in promoting their phagocytosis. CLRs are a family of transmembrane proteins that possess at least one C-type lectin-like domain (CTLD), CLRs typically have a short intracellular signaling tail that interacts with FcRs and thus mediates signaling (Li and Underhill, 2020). Mincle (macrophages-induced C-type lectin, CLEC4E) is a type II CLR family protein, belonging to the Dectin-2 subfamily of innate immune receptors. Mincle can affect phagocytosis in several ways. In general, Mincle recognizes and binds microbial surface ligands, triggering the FCRγ-Mincle Syk-CARD9 signaling pathway to promote inflammatory signaling and accelerate microbial capture and uptake (Huang et al., 2023). In addition, it may directly trigger enough intracellular signal transduction, even in the absence of other signals, to trigger bacterial cell membranes, actin reorganization and phagocytosis (Lu et al., 2018). Accumulating evidence suggests that Mincle can recognize pathogens such as bacteria, fungi and parasites, as well as endogenous ligands, and is an important PRR (pattern recognition receptor) that plays an important role in phagocytosis and elimination of pathogens (Miyaji et al., 2015). In addition, Mincle expression is tightly regulated by the NF-κB signaling pathway (Kingeter and Lin, 2012). In lipopolysaccharide (LPS)-induced macrophages, NF-κB/p65 binding to the Mincle promoter region, and blocking TLR4 (Toll-like receptors 4) or NF-κB inhibited LPS-induced expression of Mincle in macrophages.

In this study, we investigated whether PUN promotes Mincle-mediated macrophage phagocytosis via the NF-κB and MAPK signaling pathways, leading to enhanced macrophage-mediated bacterial clearance in infection. Our findings identified a new natural polyphenol compound that could enhance the phagocytosis function of macrophages and ameliorate bacterial infection, revealing a new pharmacological action and mechanism of PUN and laying the foundation for further clinical applications.