An ocular Th1 immune response promotes corneal nerve damage independently of the development of corneal epitheliopathy

Abstract

Proper sight is not possible without a smooth, transparent cornea, which is highly exposed to environmental threats. The abundant corneal nerves are interspersed with epithelial cells in the anterior corneal surface and are instrumental to corneal integrity and immunoregulation. Conversely, corneal neuropathy is commonly observed in some immune-mediated corneal disorders but not in others, and its pathogenesis is poorly understood. Here we hypothesized that the type of adaptive immune response may influence the development of corneal neuropathy. To test this, we first immunized OT-II mice with different adjuvants that favor T helper (Th)1 or Th2 responses. Both Th1-skewed mice (measured by interferon-γ production) and Th2-skewed (measured by interleukin-4 production) developed comparable ocular surface inflammation and conjunctival CD4+ T cell recruitment but no appreciable corneal epithelial changes upon repeated local antigenic challenge. Th1-skewed mice showed decreased corneal mechanical sensitivity and altered corneal nerve morphology (signs of corneal neuropathy) upon antigenic challenge. However, Th2-skewed mice also developed milder corneal neuropathy immediately after immunization and independently of ocular challenge, suggestive of adjuvant-induced neurotoxicity. All these findings were confirmed in wild-type mice. To circumvent unwanted neurotoxicity, CD4+ T cells from immunized mice were adoptively transferred to T cell-deficient mice. In this setup, only Th1-transferred mice developed corneal neuropathy upon antigenic challenge. To further delineate the contribution of each profile, CD4+ T cells were polarized in vitro to either Th1, Th2, or Th17 cells and transferred to T cell-deficient mice. Upon local antigenic challenge, all groups had commensurate conjunctival CD4+ T cell recruitment and macroscopic ocular inflammation. However, none of the groups developed corneal epithelial changes and only Th1-transferred mice showed signs of corneal neuropathy. Altogether, the data show that corneal nerves, as opposed to corneal epithelial cells, are sensitive to immune-driven damage mediated by Th1 CD4+ T cells in the absence of other pathogenic factors. These findings have potential therapeutic implications for ocular surface disorders.

CD4+T cells drive corneal nerve damage but not epitheliopathy in an acute aqueous-deficient dry eye model

“…However, the heterogeneous presentations of DED include a neuropathic form with minimal corneal epitheliopathy and ocular surface inflammation but overt symptoms and detectable corneal nerve alterations (5). In line with this, we observed in a murine tear hyperosmolarity model that corneal nerve changes can ensue in the absence of corneal barrier disruption (42), and more recently, that corneal neuropathy may develop independently of corneal epitheliopathy if a type 1 immune response is present in the ocular surface (43). However, since both models lack ocular desiccation, the defining disease feature that initiates corneal pathology (3,4), their conclusions may not apply to DED because additional mechanisms could be at play.…”

“…Corneal fluorescein uptake was measured as previously described (43,47). In brief, 0.5 μl of dextran-fluorescein isothiocyanate (average molecular weight 3000-5000, 10 mg/ml in PBS) was applied to each eye and then the mouse was returned to its cage.…”

“…We have previously shown that antigen-driven CD4 + T cell activation in an ocular surface that is not exposed to desiccation is sufficient to cause corneal nerve damage (43). However, other factors may also determine whether corneal nerve damage occurs in the context of an ocular surface immune response since CD4 + T cell activation drives corneal nerve damage in herpetic keratitis and ocular graft-versus-host disease but not in allergic conjunctivitis mouse models (44).…”

“…Since corneal mechanosensitivity reduction in mouse DED models correlates with structural changes (38,39), we evaluated corneal nerve morphology on day 10 (Figures 2F-I and Supplementary Figure 2E) at three different levels within the intraepithelial innervation (43,47). DED WT mice had reduced corneal nerve density at the subapical, mid-epithelial, and subbasal levels.…”

“…Our results so far supported our hypothesis that the adaptive immune response drives corneal nerve damage in DED. Based on our previous observation that isolated ocular CD4 + T cell activation is capable of inducing corneal neuropathy (43), we hypothesized that the lack of CD4 + T cells in Rag1KO mice was responsible for their resistance to develop corneal nerve damage in the context of DED. To test this, we isolated splenic CD4 + T cells from control or DED WT mice 10 days after surgery and transferred them into sex-matched naïve Rag1KO mice, which were then assessed weekly for DED pathology (Figure 5A).…”