title: In vivo downregulation of innate and adaptive immune responses in corneal allograft rejection by HC-HA/PTX3 complex purified from amniotic membrane.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4(+) T cells.
authors: TissueTech, Inc., Miami, Florida.
authors: Hua
authors: H
authors: He
authors: Yaohong
authors: Y
authors: Tan
authors: Stephanie
authors: S
authors: Duffort
authors: Victor L
authors: VL
authors: Perez
authors: Scheffer C G
authors: SC
authors: Tseng
mesh_terms: D000209:Acute-Phase Proteins; D056704:Adaptive Immunity / Q000502:physiology*; D064591:Allografts; D000650:Amnion / Q000166:cytology / Q000276:immunology*; D000818:Animals; D002097:C-Reactive Protein / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D016039:Corneal Transplantation; D016207:Cytokines / Q000096:biosynthesis / Q000187:drug effects; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006084:Graft Rejection / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D007113:Immunity, Innate / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D000683:Serum Amyloid P-Component / Q000494:pharmacology*; D000099153:Pentraxins
chemical_list: D000209:Acute-Phase Proteins; D002097:C-Reactive Protein; D016207:Cytokines; D006820:Hyaluronic Acid; D000683:Serum Amyloid P-Component; D000099153:Pentraxins
keywords: CD4+ T cell; amniotic membrane; corneal allograft rejection; macrophage polarization