Effect of molecular weight of tyramine-modified hyaluronan on polarization state of THP-1 and peripheral blood mononuclear cells-derived macrophages

The immunomodulatory properties of hyaluronan and its derivatives are key to their use in medicine and tissue engineering. In this work we evaluated the capability of soluble tyramine-modified hyaluronan (THA) synthesized from hyaluronan of two molecular weights (low M_w = 280 kDa and high M_w = 1640 kDa) for polarization of THP-1 and peripheral blood mononuclear cells (PBMCs)-derived macrophages (MΦs). We demonstrate the polarization effects of the supplemented THA by flow cytometry and bead-based multiplex immunoassay for the THP-1 derived MΦs and by semi-automated image analysis from confocal microscopy, immunofluorescent staining utilizing CD68 and CD206 surface markers, RT-qPCR gene expression analysis, as well as using the enzyme-linked immunosorbent assay (ELISA) for PBMCs-derived MΦs. Our data indicate that supplementation with LMW THA drives changes in THP-1 derived MΦs towards a pro-inflammatory M1-like phenotype, whereas supplementation with the HMW THA leads to a more mixed profile with some features of both M1 and M2 phenotypes, suggesting either a heterogeneous population or a transitional state. For cells directly sourced from human patients, PMBCs-derived MΦs, results exhibit a higher degree of variability, pointing out a differential regulation of factors including IL-10 and CD206 between the two cell sources. While human primary cells add to the clinical relevance, donor diversity introduces wider variability in the dataset, preventing drawing strong conclusions. Nevertheless, the MΦs profiles observed in THP-1 derived cells for treatments with LMW and HMW THA are generally consistent with what might be expected for the treatment with non-modified hyaluronans of respective molecular weights, confirming the known association holds true for the chemically tyramine-modified hyaluronan. We stipulate that these responses will provide basis for more accurate in vivo representation and translational immunomodulatory guidance for the use of THA-based biomaterials to a wider biomaterials and tissue engineering communities.