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Hua He, Yaohong Tan, Victor Perez, Scheffer Tseng; HC-HA Suppresses Inflammatory and Immune Responses and Improves Murine Corneal Allograft Survival. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2059.
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Experimental and clinical studies have shown that amniotic membrane (AM), AM extract, and HC-HA [a covalent complex formed by heavy chain (HC) of inter-α-trypsin inhibitor (IαI) and hyaluronan (HA)] suppress pro-inflammatory responses. Hence, we decide to determine whether HC-HA can regulate T cell responses and reduce murine corneal allograft rejection.
T cell activation was assessed by cell proliferation and cytokine production in splenocytes from Ova T cell receptor transgenic mice. Optimization of injection sites, volume, and frequency with HC-HA before or during intracorneal injection of LPS was determined by influx of EGFP+ macrophages into corneas of Mafia mice. At day 4 after HC-HA treatment, corneas were digested with 820 units/ml of collagenase at 37 °C for 1 h. EGFP- and EGFP+ cells were isolated by FACS. mRNA expression of Arg-1, IL-10, and IL-12 was measured by qPCR. Allogeneic corneal transplantation was performed using wild-type BALB/c mice as recipients and C57BL/6 mice as donors, and its outcome scored by graft clarity measured twice a week using slit lamp biomicroscopy. Grafts that received two consecutive scores ≥ 3 without resolution were considered rejected.
HC-HA but not HA at 1 mg/ml significantly suppressed the proliferation and production of IFN-γ and IL-2 in splenocytes with OVA peptide (0 -10 μM) at day 2 and day 4 (all p < 0.05). The injection regimen was optimized by giving 5 μl at each injection between subconjunctiva and fornix to all four quadrants. Pretreatment of HC-HA 3 days prior to LPS injection significantly suppressed the influx of EGFP+ macrophages to LPS-insulted corneas (9.1±0.3 vs.12.3±0.4, HC-HA vs PBS, p =0.02). Importantly, even though EGFP+ macrophages did migrate into corneas, some of them were polarized into M2 phenotype as suggested by significant up-regulation of Arg-1 and IL-10 but down-regulation of IL-12 (p < 0.05). Compared to PBS control, allograft rejection was significantly suppressed by injection of 10 μl HC-HA at one quadrant twice a week (p < 0.05), and further reduced by injection with 5 μl at 4 quadrants twice a week (p < 0.002).
HC-HA significantly suppresses murine corneal allograft rejection. The mechanism of this action may be contributed by HC-HA’s ability to down-regulate pro-inflammatory macrophages and to suppress T cell immune response.
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