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Sean Tighe, Hua He, Suzhen Zhang, Scheffer Tseng; Polarization of Anti-inflammatory M2 Macrophages by HC-HA Complex Purified from Amniotic Membrane or Reconstituted In Vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2060. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Native HC-HA [a covalent complex formed by heavy chain (HC) of inter-α-trypsin inhibitor (IαI) with hyaluronan (HA)] purified from human amniotic membrane (AM) possesses anti-inflammatory actions by polarizing M2 macrophages and curtailing T cell activation. Herein, we further delineate this anti-inflammatory role by examining HA complex formed by in vitro reconstitution with different components, i.e., HA, HC, and PTX3, with or without the catalytic action of TSG-6.
High molecular weight HA was covalently immobilized on CovaLink NH 96 wells using Sulfo-NHS and EDAC. Purified IαI and recombinant PTX3 and TSG-6 were then added in different sequences to form different HA complexes, of which the composition was characterized by ELISAs and Western blotting. Their anti-inflammatory actions were assessed in IFN-γ/LPS stimulated RAW264.7 cells by qPCR and ELISAs to quantify mRNA and protein expression of IL-10, IL-12, and IL-23.
Only PTX3, but not TSG-6, could be released by hyaluronidase digestion or a mild alkaline treatment with 50 mM NaOH in native HC-HA purified from AM. HA complexes, formed by either PTX3 or TSG-6, were resistant to extraction by 6M guanidine HCl. TSG-6 dose-dependently inhibited PTX3 binding but not vice versa. Transfer of HCs from IαI to HA only occurred in the presence of TSG-6 but not PTX3. IFN-γ/LPS stimulated RAW264.7 cells on immobilized native HC-HA, but not on immobilized HA alone, expressed significantly higher IL-10 but undetectable IL-12 and IL-23. HA complex with TSG-6 or HC/TSG-6 suppressed IL-12 expression but did not induce IL-10, while HA complex with PTX3 induced IL-10 expression and suppressed IL-12 expression. Neither of the above conditions was able to suppress IL-23. Only HC-HA complex formed by sequential addition of PTX3 followed by IαI/TSG-6 inhibited IL-23.
PTX3 is an essential component in native HC-HA purified from AM. The anti-inflammatory action of native HC-HA can be recapitulated in vitro by step-wise addition of PTX3 followed by IαI/TSG-6 to HA. Such information is useful for future scale-up manufacturing and dissection of the molecular mechanism of AM’s anti-inflammatory action.
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