June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
HC-HA Suppresses Inflammatory and Immune Responses and Improves Murine Corneal Allograft Survival
Author Affiliations & Notes
  • Hua He
    TissueTech. Inc., Miami, FL
  • Yaohong Tan
    Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL
  • Victor Perez
    Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL
    Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL
  • Scheffer Tseng
    TissueTech. Inc., Miami, FL
    Ocular Surface Center, and Ocular Surface Research Education Foundation, Miami, FL
  • Footnotes
    Commercial Relationships Hua He, TissueTech, Inc. (E); Yaohong Tan, None; Victor Perez, Alcon (C), Bausch & Lomb (C), Genentech (C), Cleveland Clinic Foundation (P), Alcon (F), Alcon (R); Scheffer Tseng, NIH, NEI (F), TissueTech, Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2059. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Keywords: 557 inflammation • 656 protective mechanisms • 741 transplantation  
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