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.

Resident corneal c-fms + macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis

“…Prior studies demonstrated unequivocally the capacity of cultured keratocytes to express biologically significant quantities of CXCL8, prior to the expression of other chemokines tested (Natarajan et al 2003). We also showed in an in vitro 3-dimensional model of the human cornea that in the absence of any other cell type, the presence of keratocytes is sufficient for infiltration of peripheral blood leukocytes upon HAdV-D37 infection (Rajaiya et al 2015). We also demonstrated previously using CXCL1 −/− and CXCR2 −/− mice that deficiency of either CXCL1 or its receptor resulted in delayed infiltration of neutrophils, but not reduced numbers of inflammatory monocytes in HAdV-D37 corneal infection (Chintakuntlawar and Chodosh 2009).…”

New Paradigms for the Study of Ocular Alphaherpesvirus Infections: Insights into the Use of Non-Traditional Host Model Systems

“…Corneal facsimiles, comprised of cornea cells grown in matrices, have sought to mimic the 3D structure of the cornea and cell-to-cell interactions in a renewable and cost-effective manner. One such model consists of human corneal stromal fibroblasts mixed with type I collagen in transwell plates overlaid with Matrigel to simulate the corneal stroma and basement membrane and this model has been used for corneal inflammation and adenovirus infection studies [ 91 ]. Subsequent improvements consisted of culturing epithelial cells over these stromal cells and the incorporation of an endothelium to create full thickness corneal equivalents [ 92 ].…”

Genomic foundations of evolution and ocular pathogenesis in human adenovirus species D

“…Matrigel is a gelatinous protein complex secreted by cultured mouse sarcoma cells, containing many components of epithelial basement membrane, and previously shown to bind interferon gamma . Immunohistochemistry performed on facsimiles overlaid with Matrigel and after overnight infection with HAdV‐D37 showed foci of CXCL8 bound to the Matrigel, and colocalized to heparan sulfate . Heparan sulfate is a component of corneal epithelial basement membrane also present in Matrigel, and expected to bind to CXCL8 because of its negative charge .…”