Epithelial Dendritic Cells and Subbasal Nerve Plexus in Infectious Keratitis: An In Vivo Confocal Microscopy Study. Andrea Cruzat, MD, Dimos Mantopoulos, MD, Kristina Kurbanyan, MD, Lixin Zheng, MD Pedram Hamrah, MD.  Cornea Service & Ocular Surface Imaging Center, Massachusetts Eye & Ear, Harvard Medical School, Boston, MA. Authors Have Non-Financial Interest Funding: NEI K12-EY016335, New England Corneal Transplant Research Fund, Falk Medical Research Trust.

Epithelial Dendritic Cells and Subbasal Nerve Plexus by In vivo confocal microscopy. Bone marrow-derived dendritic cells (DC) residing in the central cornea are typically in an immature state. Previous studies have shown that soluble factors derived from the cornea and aqueous humor, may prevent DC from acquiring phenotypic and functional maturation. Maturation of DC is induced by corneal injury, inflammation or infection. Corneal nerves are of great interest due to their important roles in regulating corneal sensation, epithelial integrity, proliferation, wound healing and for their protective functions. In the current study, we studied a possible link between the nervous system and immune cells in the cornea, by assessing the density of corneal DC and nerve alterations in infectious keratitis (IK) by laser in vivo confocal microscopy (IVCM).

Purpose: To evaluate the subbasal corneal nerve alterations and their correlation to epithelial DC density in acute IK by laser IVCM. In vivo confocal microscopy (IVCM) enables the non-invasive examination of corneal nerves and DC, comparable to ex vivo histological techniques. Dendritic cells (DC) are strategically positioned as immune sentinels and respond to invading pathogens in the cornea.

Methods: Retrospective, case-control study Methods: Retrospective, case-control study. IVCM was performed in 36 eyes with acute bacterial, acanthamoeba, fungal, and adenoviral keratitis, as well as in 20 normal eyes. IVCM of the central cornea with the Heidelberg Retina Tomograph 3 with the Rostock Cornea Module (Heidelberg Engineering GmbH). Two masked observers reviewed the in-vivo confocal images. Corneal subbasal nerve plexus density, total number of nerves, main nerve trunks and nerve branches were assessed and correlated to DC density.

Epithelial dendritic cells are visualized in the basal epithelial layer and subbasal nerve plexus in all patients. Infectious keratitis. Oblique cut of the epithelium, basal membrane, Bowman’s layer and anterior stroma. Epithelial dendritic cells shown with yellow arrows.

The corneal subbasal nerve plexus was significantly diminished in infectious keratitis compared to controls. Nerve tortuosity was significantly increased in IK compared to controls (*p<0.001) Decreased branching pattern, total nerve numbers and main trunks.(*p<0.001)

Increased dendritic cells are found with diminishment of corneal nerves in infectious keratitis. B C D Normal subbasal nerve plexus. Infectious keratitis: Decreased nerves and increased dendritic cells. C-D) Oblique cut. Subbasal nerve plexus layer with increased dendritic cells between epithelium and stroma.

Nerve density was decreased while dendritic cells were significantly higher in infectious keratitis compared to controls Mean DC density was significantly higher in all subgroups compared to controls (p<0.005). The subbasal nerve density was significantly decreased (p<0.001). Density

In infectious keratitis corneal nerve parameters were decreased while an increase in dendritic cell density was found.   Nerve Density Total Nerves Main Nerves Tortuosity Branching Dendritic cells controls 2950.08 16.2 5.3 1.6 6.0 41.3 infectious keratitis 236.9 1.5 1.0 2.5 0.5 888.3 p-Value 0.003 <0.001 0.01

Correlation between reduction in nerve density and increase in DC density in Infectious keratitis. In patients with infectious keratitis, while a decresase in corneal nerves is found, an increase of dendritic cells is observed.

In eyes with IK, DC size and processes increased over time, suggesting a more mature phenotype. B C Follow-up of acanthamoeba keratitis at 1 week (A), 5 weeks (B) and 8 weeks (C). Lack of subbasal nerve plexus and phenotypic evolution of dendritic cells, increasing in size and dendritic processes.

In summary, the the diminishment of subbasal corneal nerves was associated with an increase in dendritic cell density in infectious keratitis. Laser IVCM revealed the presence of central corneal DC in all patients and controls, increasing significantly in the presence of infection. The increase in DC associated with the decreased subbasal corneal nerves plexus, suggesting a possible direct interaction between the immune system and the nervous system in the cornea. Further studies are needed to determine whether and how corneal nerves regulate DC in the cornea.