Human macrophages release higher IL-1ra over IL-1beta when stimulated by block acetylated chitosan microparticles and not by random acetylated chitosans or water-soluble oligomers

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

Mots clés

• Regenerative Medicine
• cytokine
• Biocompatibility
• polymer