Understanding the inflammatory effect of hyaluronic acid and hyaluronic acid coated nanoparticles in THP-1 macrophages

Abstract

Hyaluronic acid (HA) is known to induce both pro- and anti-inflammatory effects when interacting with cell receptors such as CD44 and TLR. In particular, the molecular weight of soluble HA is supposed to induce different inflammatory responses. Low-molecular-weight HA (100 – 500 kDa) are reported to induce pro- inflammatory responses, whereas high-molecular-weight HA (> 1000 kDa) should promote anti-inflammatory responses. In this research, soluble hyaluronic acid and chitosan/hyaluronic acid nanoparticles were investigated to assess their potential anti-inflammatory effect on CD44-expressing cells. Because of the nanoparticles’ size and HA surface decoration, it was believed that the nanoparticles may interact with CD44 receptors in a similar way as high-molecular-weight HA. Therefore, CS/HA nanoparticles are hypothesized to have an anti-inflammatory effect like high-molecular-weight HA. To study the effects of HA and identify key features for alternative HA-based treatments, the THP-1 cell line was selected for the study. Initially, a protocol to differentiate and polarize THP-1 in three sub-types macrophages was developed. THP-1 macrophages were polarized to uncommitted (M0), pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. Macrophages were characterized by morphology, cytokine expression, marker expression and gene expression of the cells. THP-1 macrophages were incubated with soluble HA and CS/HA nanoparticles for 24 hours and with different concentrations. After the incubation, the expression of pro-inflammatory cytokines, membrane-bound markers and macrophages-related genes was investigated to assess variation in macrophages. The results showed that HA of MW of 35 kDa, 111 kDa and 2,380 kDa could induce anti-inflammatory effects on THP-1 macrophages, as the expression of pro- inflammatory cytokines including TNF-ɑ and IL-1β decreased. Instead, nanoparticles prepared using low molecular weight chitosan/HA (LCS/HA) might have a pro- inflammatory effect on THP-1 macrophages, because of increased expression of pro-inflammatory cytokines, including IL-1β and GM-CSF and M1-related genes such as TNF-ɑ, IL-1β, IL-6 and CCR7. Contradictory results were obtained for soluble HA (849 kDa) and nanoparticles prepared with high molecular weight chitosan (HCS/HA): decreased expression of pro-inflammatory cytokines such as TNF-ɑ and increased expression of M1-related genes, including TNF-ɑ and CCR7, were reported. This study evidenced that it is not possible to draw a clear correlation between HA molecular weight and inflammatory processes. This observation is also mapped when investigating the effect of HA-based materials, i.e. CS/HA nanoparticles. It was not possible to detect a clear anti-inflammatory activity of nanoparticles on macrophages; additionally, LCS/HA nanoparticles tend to have an anti-inflammatory effect. Additional experiments are necessary to determine the effect of HA-based treatments, potentially looking at different timeframes or HA concentrations. In addition to considerations on the biomaterials, it can be also recommended to used different methods to differentiate THP-1 macrophages and be able to have a clear distinction between the three phenotypes of macrophages used in the study.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Katie Finegan (Supervisor), Nicola Tirelli (Supervisor), Costas Demonacos (Supervisor) & Annalisa Tirella (Supervisor)