Introduction: Human macrophages were previously shown to release excess IL-1ra over IL-1β when stimulated by chitosan microparticles (140 kDa, 80% glucosamine and 20% block N-acetyl glucosamine, 80% degree of deacetylation (DDA))[1]. These data suggest the potential of using chitosan to treat knee joint inflammation by guiding the in situ release of immunomodulatory cytokines. The purpose of this study was to identify the minimal chitosan motif required to induce higher IL-1ra release, using a U937 human macrophage model and a novel chitosan library with distinct DDA, acetylation patterns, and array of 5 different number-average molecular weights (Mn).
Materials and Methods: 15 chitosans with different Mn, DDA, and block (B) or random (R) acetylation pattern were generated, including acid-soluble ≥ 140 kDa and 10 kDa chitosans, and water-soluble 1, 3, 5 kDa oligomers (Table 1). B-acetylated (80% DDA, Mn=190 kDa) or fully deacetylated (98% DDA, Mn=140 kDa) chitosans were nitrous acid depolymerized. 98% DDA chitosans were reacetylated to 60% or 80% DDA to obtain R-acetylated chitosans. Chitosan Mn and polydispersity were determined by size exclusion chromatography and DDA by H1 NMR. Phorbol ester-differentiated U937 macrophages were stimulated for 24 hours with 5, 50 or 150 µg/mL chitosans that form particles in culture medium at an Mn ≥ 10 kDa , or with LPS and IL-4 as controls. Cell culture medium was analyzed for cytokine release by ELISA (N=3). Cell cytotoxicity was determined by lactate dehydrogenase release.
Results: Macrophages with no stimulation released high levels of IL-1ra (5364±1720 pg/mL) and negligible levels of IL-1β (56±6 pg/mL). Amongst all chitosans, only the B-acetylated 80% DDA 190 kDa chitosan (80-190K-B) stimulated a reproducible 2 to 3-fold increase in IL-1ra release at selected doses (Fig. 1A). The B-acetylated 80% DDA 10 and 190 kDa chitosans stimulated a 2 to 5-fold increase in IL-1β release (Fig. 1B). In contrast, R-acetylated 80% DDA 140 kDa (80-140K-R) chitosan induced a modest 2-fold increase in IL-1β without further enhancing IL-1ra release (Fig. 1). All other chitosans had no effects on cytokine release at any dose. Cell viability remained over 80% under all conditions.
Discussion: The failure of the 1 to 5 kDa B-acetylated chitosans to induce any cytokine release indicates that there is a minimal Mn required for chitosan to stimulate IL-1β in macrophages and suggests full cytocompatibility of chitosan oligomers at 150 µg/mL. At 80% DDA and above 10 kDa Mn, the B-acetylation pattern contributes to stimulating IL-1β release. These data are consistent with previous report suggesting that chitosan microparticle formation is necessary to activate the inflammasome and stimulate IL-1β liberation from macrophages[2]. The ability of the 80-190K-B, and not the 80-140K-R, to enhance IL-1ra release indicates that the B-acetylation pattern is necessary to shift macrophages towards a more anabolic phenotype.
Conclusion: A novel, comprehensive chitosan library was successfully generated to evaluate the impact of chitosan Mn, DDA and acetylation pattern on cytokine release in human macrophages. Water-soluble chitosan oligomers had no influence on IL-1ra and IL-1β release. Above 100 kDa, 80% DDA B-acetylated but not R-acetylated chitosans stimulated more IL-1ra, suggesting that B-acetylated chitosans are more useful for inducing the release of anti-inflammatory factors that preserve joint health.
This work was funded by the Canadian Institutes of Health Research. Salary support was from the Fonds de recherche du Québec - Santé (FRQ-S, CDH), Fonds de recherche du Québec - Nature et Technologies (FRQ-NT, DF) and the Natural Sciences and Engineering Research Council (NSERC, PGG).
References:
[1] Fong D, Ariganello MB, Girard-Lauzière J, Hoemann CD. ''Biodegradable chitosan microparticle induce delayed STAT-1 activation and lead to distinct cytokine responses in differentially polarized human macrophages in vitro''. Acta Biomaterialia 2015;12:183-94
[2] Bueter CL, Lee CK, Rathinam VA, Healy GJ, Taron CH, Specht CA, Levitz SM. ''Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis''. J. Biological Chemistry. 2011;286:35447-55