Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and anti-inflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca²⁺ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4); on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.

We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.

Cancer stem cells (CSCs) have drawn much attention as important tumour-initiating cells that may also be crucial for recurrence after chemotherapy. Although the activity of CSCs in various forms of cancer is complex and yet to be fully elucidated, opportunities for therapies targeting CSCs exist. CSCs are molecularly distinct from bulk tumour cells, so they can be targeted by exploiting their signature molecular pathways. Inhibiting stemness has the potential to reduce the risk posed by CSCs by limiting or eliminating their capacity for tumorigenesis, proliferation, metastasis, and recurrence. Here, we briefly described the role of CSCs in tumour biology, the mechanisms involved in CSC therapy resistance, and the role of the gut microbiota in cancer development and treatment, to then review and discuss the current advances in the discovery of microbiota-derived natural compounds targeting CSCs. Collectively, our overview suggests that dietary intervention, toward the production of those identified microbial metabolites capable of suppressing CSC properties, is a promising approach to support standard chemotherapy.

Introduction: Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson’s disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear.Methods: Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1 μM) for 4 h and 24 h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C–C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages.Results: Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4 h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24 h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4 h αS-exposed THP-1 cells showed increased spontaneous migration, while 24 h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia.Discussion: In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.

The anti-inflammatory activity of coffee extracts is widely recognized and supported by experimental evidence, in both in vitro and in vivo settings, mainly murine models. Here, we investigated the immunomodulatory properties of coffee extracts from green (GCE) and medium-roasted (RCE) Coffea canephora beans in human macrophages. The biological effect of GCE and RCE was characterized in LPS-stimulated THP-1-derived human macrophages (TDM) as a model of inflammation. Results showed decreased amounts of TNF-α, IL-6 and IL-1β and a strong dose-dependent inhibition of interferon-β (IFN-β) release. Molecular mechanism of IFN-β inhibition was further investigated by immunofluorescence confocal microscopy analysis that showed a diminished nuclear translocation of p-IRF-3, the main transcription factor responsible for IFN-β synthesis. The inhibition of IFN-β release by RCE and GCE was also confirmed in human primary CD14⁺ monocytes-derived macrophages (MDM). The main component of coffee extracts, 5-O-caffeoylquinic acid (5-CQA) also inhibited IFN-β production, through a mechanism occurring downstream to TLR4. Inhibition of IFN-β release by coffee extracts parallels with the activity of their main phytochemical component, 5-CQA, thus suggesting that this compound is the main responsible for the immunomodulatory effect observed. The application of 5-CQA and coffee derived-phytoextracts to target interferonopathies and inflammation-related diseases could open new pharmacological and nutritional perspectives.

Lipopolysaccharide (LPS) mimicry leading to toll-like receptor 4 (TLR4) active compounds has been so far based mainly on reproducing the lipid A portion of LPS. Our work led to a series of structurally simplified synthetic TLR4 agonists in preclinical development as vaccine adjuvants called FPs. FPs bind MD2/TLR4 similarly to lipid A, inserting the lipid chains in the MD2 lipophilic cavity. A strategy to improve FPs' target affinity is introducing a monosaccharide unit in C6, mimicking the first sugar of the LPS core. We therefore designed a panel of FP derivatives bearing different monosaccharides in C6. We report here the synthesis and optimization of FPs' C6 glycosylation, which presented unique challenges and limitations. The biological activity of glycosylated FP compounds was preliminarily assessed in vitro in HEK-Blue cells. The new molecules showed a higher potency in stimulating TLR4 activation when compared to the parent molecule while maintaining TLR4 selectivity.