title: Novel model of innate immunity in corneal infection.
abstract: The cornea functions as the major refractive interface for vision and protects the internal eye from insult. Current understanding of innate immune responses to corneal infection derives from a synthesis of in vitro and in vivo analyses. However, monolayer cell cultures and mouse models do not accurately duplicate all aspects of innate immunity in human patients. Here, we describe a three-dimensional culture system that incorporates human cells and extracellular matrix to more completely simulate the human cornea for studies of infection. Human corneal stromal fibroblasts were mixed with type I collagen in 3-μm pore size transwell inserts, and overlayed with Matrigel to simulate a human corneal stroma and epithelial basement membrane. These were then infected with a cornea-tropic adenovirus, and exposed on their inferior side to leukocytes derived from human peripheral blood. Subsequent analyses were performed with histology, confocal microscopy, ELISA, and fluorescence-activated cell sorting (FACS). CXCL8, a neutrophil chemokine shown previously as the first cytokine induced in infection of human corneal cells, increased upon adenovirus infection of facsimiles in a dose-responsive fashion. Myeloperoxidase-positive cells infiltrated infected corneal facsimiles in a sub-Matrigel location, possibly due to CXCL8 colocalization with heparan sulfate, a Matrigel constituent. Cellular infiltration was significantly inhibited by treatment with chemical inhibitors of p38 MAPK and Src kinase, both constituents of a signaling cascade previously suggested to regulate inflammation after adenovirus infection. FACS analysis determined that both virus and corneal fibroblasts were necessary for the induction of leukocyte migration into the facsimiles. The corneal facsimile, literally a cornea in a test tube, permits mechanistic studies on human tissue in a highly tractable system.
authors.affiliation: Howe Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA.
authors.forename: Jaya
authors.initials: J
authors.lastname: Rajaiya
authors.affiliation: Howe Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Howe Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Barequet
authors.affiliation: Howe Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Gilmore
authors.affiliation: Howe Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, 243 Charles Street, Boston, MA, 02114, USA. james_chodosh@meei.harvard.edu.|Massachusetts Eye and Ear Infirmary, Howe Laboratory, 243 Charles Street, Boston, MA, 02114, USA. james_chodosh@meei.harvard.edu.
authors.forename: James
authors.initials: J
authors.lastname: Chodosh
mesh_terms: D000258:Adenovirus Infections, Human / Q000276:immunology; D001485:Basement Membrane / Q000276:immunology; D003315:Cornea / Q000166:cytology / Q000276:immunology / Q000821:virology; D003316:Corneal Diseases / Q000276:immunology* / Q000821:virology; D060527:Corneal Keratocytes / Q000276:immunology / Q000502:physiology; D003319:Corneal Stroma / Q000166:cytology / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology*; D018613:Microscopy, Confocal; D018448:Models, Immunological
keywords: Adenovirus; In vitro model; Keratitis