The effects of the molecular weights of hyaluronic acid on the immune responses.

Background: The molecular weight of hyaluronic acid (HyA) depends on the type of organ in the body. When HyA of the desired molecular weight is implanted into the human body for regeneration of damaged tissue, it is degraded by hyaluronidase in associated with an inflammatory response. This study sought to evaluate the effects of HyA molecular weight and concentration on pro- and anti-inflammatory responses in murine macrophages.

Methods: The structures and molecular weights of HyAs (LMW-10, MMW-100, MMW-500, and HMW-1,500) were confirmed by  H NMR and gel permeation chromatography (GPC), respectively. After treatment of murine macrophages with a low (10 µg/mL) or high (100 µg/mL) concentration of each molecular weight HyA, cells were stimulated with lipopolysaccharide (LPS) and changes in immune response in both LPS-stimulated and untreated macrophages were evaluated by assessing nitric oxide (NO) production, and analyzing expression of pro- and anti-inflammatory genes including by RT-PCR.

Results: Molecular weights of LMW-10, MMW-100, MMW-500, and HMW-1,500 were 13,241 ± 161, 96,531 ± 1,167, 512,657 ± 8,545, and 1,249,500 ± 37,477 Da, respectively. NO production by LPS-stimulated macrophages was decreased by increasing concentrations and molecular weights of HyA. At a high concentration of 100 µg/mL, HMW-1,500 reduced NO production in LPS-stimulated macrophages to about 45 %. Using NanoString technology, we also found that the immune-related genes TNF-α, IL-6, IL-1β, TGF-β1, IL-10, IL-11, CCL2, and Arg1 were specifically over-expressed in LPS-stimulated macrophages treated with various molecular weights of HyA. An RT-PCR analysis of gene expression showed that HMW-1,500 decreased expression of classically activated (M1) macrophage genes, such as TNF-α, IL-6, CCL2, and IL-1β, in LPS-stimulated macrophages, whereas medium molecular-weight HyA (MMW-100 and MMW-500) instead increased expression levels of these genes. HMW-1,500 at a high concentration (100 µg/mL) significantly decreased expression of pro-inflammatory genes in LPS-stimulated macrophages. Expression of genes associated with anti-inflammatory responses (M2 phenotype), such as TGF-β1, IL-10, IL-11, and Arg1, were increased by high concentrations of MMW-500 and HMW-1,500 in LPS-stimulated macrophages.

Conclusions: High molecular-weight HyA (i.e., > 1,250 kDa) inhibits pro-inflammatory responses in LPS-stimulated macrophages and induces anti-inflammatory responses in a concentration dependent manner.