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Hua He, Yaohong Tan, Stephanie Duffort, Victor L. Perez, Scheffer C. G. Tseng; In Vivo Downregulation of Innate and Adaptive Immune Responses in Corneal Allograft Rejection by HC-HA/PTX3 Complex Purified From Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2014;55(3):1647-1656. doi: 10.1167/iovs.13-13094.
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Heavy chain–hyaluronic acid (HC-HA)/PTX3 purified from human amniotic membrane (AM) was previously observed to suppress inflammatory responses in vitro. We now examine whether HC-HA/PTX3 is able to exert a similar effect in vivo, using murine models for keratitis and corneal allograft rejection.
The in vitro effect of HC-HA/PTX3 was tested using OTII ovalbumin (OVA) transgenic, purified CD4+ T cells, or IFN-γ/lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cytokine production was measured by ELISA, while cell surface markers and cell proliferation were determined by flow cytometry. In vivo effects of HC-HA/PTX3 were analyzed by quantifying the recruitment of enhanced green fluorescence–labeled macrophages and by measuring the expression of arginase 1 (Arg-1), IL-10, and IL-12 in LPS-induced keratitis in the macrophage Fas-induced apoptosis (Mafia) mouse. The effect of corneal allograft survival in a complete major histocompatibility complex (MHC) mismatched mouse model was assessed by grading corneal opacification.
In vitro studies demonstrated that HC-HA/PTX3 significantly enhanced the expansion of FOXP3 T cells and suppressed cell proliferation and protein expression of IFN-γ, IL-2, CD25, and CD69 in activated CD4+ T cells. Furthermore, immobilized HC-HA/PTX3 significantly upregulated IL-10 gene expression but downregulated that of IL-12 and IL-23 in activated RAW264.7 cells. Finally, in vivo subconjunctival injection of HC-HA/PTX3 significantly prolonged corneal allograft survival, suppressed macrophage infiltration, and promoted M2 polarization by upregulating Arg-1 and IL-10 but downregulating IL-12.
HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4+ T cells.
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