Exploring glycopolymers targeted toward the CR domain of CD206 as functional macromolecules: synthesis and anti-inflammatory potential

The term ‘inflammation’ refers to physiological and pathological processes activated by noxious stimuli, such as tissue damage or infection. In its initial phase, Pattern Recognition Receptors (PRRs) play a pivotal role, recognizing structures called Pathogen or Damage Associated Molecular Patterns (PAMPs or DAMPs). An example of PRR is the Mannose Receptor (CD206), involved in antigen presentation, phagocytosis and signaling, potentially contributing to the development of autoimmune disorders (rheumatoid arthritis, RA), and autoinflammatory diseases (gout, pseudogout). RA is a chronic disease characterized by inflammation of the synovial joints. Recent studies have confirmed the ability of CD206 to bind the mannosylated enzyme myeloperoxidase (MPO), released by neutrophils in inflammatory conditions, like RA. Once internalized, MPO can react with H2O2 producing reactive oxygen species and pro-inflammatory cytokines, which are mediators of the disease. Gout and pseudo-gout are arthritises caused by the deposition of mono-sodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals within the joints. These crystals, recognized as DAMPs by PPRs, initiate the assembly and activation of the NLRP3 inflammasome, responsible for the production of IL-1β. This Ph.D. project aimed at developing a novel class of synthetic glycopolymers, formed by galactose units sulfated in position 3, referred as to 3-O-sulfo-galactosylated glycopolymers, capable of binding the CR domain of CD206, reversibly blocking its endocytic activity by temporarily trapping the receptor inside endosomes. The hiding of the receptor from the membrane could potentially prevent CD206-mediated endocytosis of pro-inflammatory molecules such as MPO, interrupt the signaling associated with the recognition of DAMPs like MSU and CPPD crystals and the consequent release of pro-inflammatory mediators. Active 3-O-sulfo-galactosylated and inactive galactosylated glycopolymers were synthesized by Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. The glycopolymers were tested to confirm their biocompatibility. 3-O-sulfo galactosylated glycopolymer ability to block CD206 recycling was tested on Bone Marrow-Derived Macrophages (BMDMs) phenotypes M0 and M2, expressing different levels of CD206. A non-relevant blocking was observed on M0 macrophages, while reaching 54% blocking on M2 macrophages after 2h incubation with the glycopolymer. The anti-inflammatory properties were evaluated in M0, M1 and M2 BMDMs phenotypes stimulated with MPO, in the presence or absence of 3-O-sulfo-galactosylated glycopolymer or its negative control, detecting a decrease in the production of reactive oxygen species that was more intense in cells expressing higher levels of CD206. A reduction of IL-1β production was observed for both M1 and M2 macrophages (33% and 30%, respectively). Glycopolymers were tested also in in vitro models of gout and pseudo-gout using PMA-polarized THP-1 macrophages stimulated with MSU or CPPD crystals, respectively. A decrease in the production of both intracellular and released IL-1β and CXCL1 titers was observed when cells were pre-incubated with 3-O-sulfo-beglycopolymers before stimulation with MSU crystals. However, no evident reduction of these titers was observed after stimulation with CPPD crystals. The specificity of the interaction was demonstrated by the lack of activity of the inactive galactosylated glycopolymer. Finally, the two glycopolymers were tested on an in vivo model of collagen-induced arthritis (CIA), showing an acceptable anti-inflammatory activity of 3-O-sulfo-glycopolymers, which was able of maintaining the average CIA score at lower levels over time, if compared to the controls. The treatment with 3-O-sulfo-galactosylated glycopolymer led to a remarkable decrease in anti-collagen IgG2b and IgG2c antibodies levels after 19 days of treatment, which were correlated to reduced degradation of the ankle cartilage.