Polysaccharides from the fungus Inonotus obliquus activate macrophages into a tumoricidal phenotype via interaction with TLR2, TLR4 and Dectin-1a

Background: Tumor-associated macrophages (TAMs) may both promote and suppress tumor development. Therefore, molecules that are able to activate and repolarize these cells into a tumoricidal phenotype could be of great interest as a new strategy for cancer immunotherapy. Fungal beta-glucans have been suggested as a promising way of activating TAMs, but most of the research has been carried out on particulate beta-glucans of large sizes (> 500 kDa), which potentially have different biological properties than smaller, water-soluble molecules with similar structures. Methods: Bone marrow-derived mouse macrophages were treated with 6 different purified polysaccharides isolated from the medicinal fungus Inonotus obliquus. Nitric oxide concentration was quantified using the Griess assay and by qPCR of iNOS mRNA. IL-6 and TNF-alpha concentrations were quantified using Luminex ELISA technology (using human monocyte-derived macrophages and mouse bone-marrow derived macrophages). Growth inhibition of cancer cells was measured using radiolabeled thymidine. Receptor interaction was determined using HEK-Blue reporter cell lines and TLR4 KO macrophages. Main findings: The acidic, water-soluble polysaccharides AcF1, AcF2 and AcF3 induced nitric oxide (NO) production by mouse macrophages when combined with IFN-alpha, leading to a strong subsequent tumoricidal activity by the macrophages. Tumoricidal activity of AcF1 and AcF3 was fully retained in TLR4 knockout macrophages, demonstrating that the macrophage activation was not dependent on TLR4. Further, AcF3 induced high levels of the pro-inflammatory cytokines IL-6 and TNF-alpha in human and mouse macrophages, independent of co-activation with IFN-gamma. The polysaccharides were shown to bind TLR2, TLR4 and Dectin-1a to varying degrees, and these receptors were likely to be responsible for the macrophage activation. The acidic polysaccharides AcF1, AcF2 and AcF3 strongly activated TLR2, while AcF3 and AcF1 activated TLR4. The acidic polysaccharides had low affinity to Dectin-1a compared to the polysaccharides IWN, EWN and A1, which suggests that this receptor is not the main receptor for the pro-inflammatory activity observed. Conclusion: For the first time, this study demonstrates that I. obliquus polysaccharides are able to bind multiple pattern recognition receptors to activate macrophages into a pro-inflammatory anti-tumor phenotype. The induction of tumoricidal activity in the macrophages as well as the interaction with TLR2, TLR4 and Dectin-1a suggest that the I. obliquus polysaccharides may have unique ways of interacting with macrophages, which could open up for new treatment options in cancer immunotherapy.