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-γproduction) 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.…”

“…By contrast, the resistance of corneal nerves to experimental desiccation in the absence of CD4 + T cells indicates that corneal neuropathy in DED is mostly immune-mediated. In line with this, we have previously shown that corneal nerves are particularly sensitive to the activation of Th1 CD4 + T cells in the ocular surface in the absence of desiccating stress(43), and on the other hand, that signaling through transient receptor potential vanilloid-1 channels in the corneal tissue is required to propagate desiccation-initiated corneal nerve damage in DED(47). Therefore, it is likely that in the context of DED, sustained levels of Th1 cytokines from CD4 + T cells prime corneal nerve changes that later propagate proximally through overactivation of transient receptor potential vanilloid-1 channels.…”

“…Considering the intimate relationship between corneal epithelial cells and nerve fibers, it is striking that desiccation-induced pathologic changes in the former may occur while the latter remain unaffected in the absence of CD4 + T cells. However, we have found that the converse is also true: corneal nerves may be damaged by CD4 + T cell activation in the ocular surface while the corneal epithelium remains unaffected in the absence of desiccating stress(43). Thus, the present findings extend the notion of an epithelial-neural divide in corneal pathology to DED disease.…”

“…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 Rag1 KO 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 Rag1 KO mice, which were then assessed weekly for DED pathology (Figure 5A).…”

“…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.…”