IMMUNOMODULATORY EFFECTS OF GLYCATED CHITOSAN ON MACROPHAGE PHENOTYPE AND CYTOKINE REGULATION

Abstract

Immunostimulants are a class of therapeutics focusing on enhancing or modulating the immunesystem to defend against various viruses and diseases. In the past two decades, immunostimulants have gained traction, specifically in cancer immunotherapy. N- dihydrogalactochitosan, commonly called Glycated Chitosan (GC) is a novel immunostimulant with the potential to modulate the Tumor Microenvironment (TME). Tumor-Associated Macrophages (TAMs) play a crucial role in shaping the TME, often promoting immunosuppression and resistance to cancer therapies. This is largely due to the abundance of M2-polarized TAMs that fuel tumor growth and treatment resistance. Previous literature has identified that GC can influence the TME, but its direct effects on macrophages have been largely unexplored. Here, we investigate the effects of GC on murine Bone Marrow Derived Macrophages (BMDM) polarization and inflammatory signals using in vitro murine models. Using ELISAs, surface staining, and intracellular flow cytometry, we characterized macrophage populations to assess changes in inflammatory signaling pathways and polarization post-GC treatment. Following GC stimulation of BMDMs, M0 macrophages expressed a significant increase of pro-inflammatory cytokines such as IL-1β and TNF-α. Independently, these same M0 macrophages also expressed a decrease in anti-inflammatory cytokines such as IL-10. These patterns were present in M1 and M2 macrophages as well to a lesser extent. This suggested that GC could shift macrophage phenotype towards M1 classically activated macrophages, and away from M2 alternatively activated macrophages. Individual cytokine analysis data was confirmed with flow cytometry, where GC-treated BMDMs had a greater percentage of CD86 (a pro- inflammatory macrophage marker) and a lower percentage of CD206 (an anti-inflammatory macrophage marker). Combined with global cytokine secretion multiplex data which displayed the ability of GC to upregulate the secretion of many pro-inflammatory pathways, these findings show that GC can promote macrophage polarization towards an M1-like phenotype. The observed immunomodulatory effects provide mechanistic insights into how GC can remodel the TME and support its use in cancer immunotherapy.