Thrombospondin-1-deficient (TSP-1^−/−) mice are used as an animal model of Sjögren’s Syndrome because they exhibit many of the symptoms associated with the autoimmune type of dry eye found in primary Sjögren’s Syndrome. This type of dry eye is linked to the inflammation of the lacrimal gland, conjunctiva, and cornea, and is thought to involve dysfunction of the complex neuronal reflex arc that mediates tear production in response to noxious stimuli on the ocular surface. This study characterizes the structural and functional changes to the corneal nerves that are the afferent arm of this arc in young and older TSP-1^−/− and wild type (WT) mice. The structure and subtype of nerves were characterized by immunohistochemistry, in vivo confocal microscopy, and confocal microscopy. Cytokine expression analysis was determined by Q-PCR and the number of monocytes was measured by immunohistochemistry. We found that only the pro-inflammatory cytokine MIP-2 increased in young corneas of TSP-1^−/− compared to WT mice, but tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) all increased in older TSP-1^−/− mouse corneas. In contrast, CD11b+ pro-inflammatory monocytes did not increase even in older mouse corneas. Calcitonin gene-related peptide (CGRP)-, but not Substance P (SubP)-containing corneal nerves decreased in older, but not younger TSP-1^−/− compared to WT mouse corneas. We conclude that CGRP-containing corneal sensory nerves exhibit distinct structural deficiencies as disease progresses in TSP-1^−/− mice, suggesting that: (1) TSP-1 is needed for the development or repair of these nerves and (2) impaired afferent corneal nerve structure and hence function may contribute to ocular surface dysfunction that develops as TSP-1^−/− mice age.

Diabetes mellitus (DM) is one of the most prevalent diseases globally, and its prevalence is rapidly increasing. Most patients with a long-term history of DM present with some degree of keratopathy (DK). Despite its high incidence, the underlying inflammatory mechanism of DK has not been elucidated yet. For further insights into the underlying immunopathologic processes, we utilized streptozotocin-induced mice to model type 1 DM (T1D) and B6.Cg-Lepob/J mice to model type 2 DM (T2D). We evaluated the animals for the development of clinical manifestations of DK. Four weeks post-induction, the total frequencies of corneal CD45⁺CD11b⁺Ly-6G⁻ myeloid cells, with enhanced gene and protein expression levels for the proinflammatory cytokines TNF-α and IL-1β, were higher in both T1D and T2D animals. Additionally, the frequencies of myeloid cells/mm² in the sub-basal neural plexus (SBNP) were significantly higher in T1D and T2D compared to non-diabetic mice. DK clinical manifestations were observed four weeks post-induction, including significantly lower tear production, corneal sensitivity, and epitheliopathy. Nerve density in the SBNP and intraepithelial terminal endings per 40x field were lower in both models compared to the normal controls. The findings of this study indicate that DM alters the immune quiescent state of the cornea during disease onset, which may be associated with the progressive development of the clinical manifestations of DK.

After transplantation, Th1-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b ⁺ DCs (DC2) and CD103 ⁺ DCs (DC1). This interaction necessitates further investigation in the context of transplant immunity. We used well-established preclinical models of corneal transplantation and identified distinct roles of migratory CD103 ⁺ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103 ⁺ DC1 demonstrate a tolerogenic phenotype that modulates the immunogenic capacity of CD11b ⁺ DC2 primarily mediated by IL-10, suppressing alloreactive CD4 ⁺ Th1 cells via the PD-L1/PD-1 pathway and enhancing Treg-mediated tolerance via αvβ8 integrin–activated TGF-β1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4 ⁺ Th1 cells induced migratory CD103 ⁺ DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers, including αvβ8 integrin and IL-10, and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex vivo induced tolerogenic CD103 ⁺ DC1 (iDC1) effectively inhibited Th1 polarization and preserved the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103 ⁺ DC1 in modulating host alloimmune responses.

Objectives: In this study, we establish a protocol for evaluating the outcomes of endothelial keratoplasty, including graft survival, rejection, or failure. Additionally, we also evaluate the alloimmune response in graft recipients. Methods: We performed EK using C57BL/6 (allogeneic) and BALB/c (syngeneic) as donors and BALB/c mice as recipients. Slit-lamp examination and optical coherence tomography were performed for clinical evaluations for 16 weeks post-procedure. Criteria for the assessment of corneal opacity were established and the animals were graded weekly. Additionally, we assessed corneal endothelial cell density by harvesting the corneas and staining with zonula occludens-1 (ZO-1). Lastly, lymph nodes were collected, and CD4⁺ T cells were MACS-sorted and co-cultured with syngeneic or allogeneic antigen-presenting cells (APCs) to assess the IFN-γ expression levels by alloreactive Th1 cells (ELISPOT) in response to the direct (donor) or indirect (host) pathways of sensitization. Results: We observed graft failure in four animals, including irreversible corneal opacity, graft detachment, and anterior synechiae in the first four weeks. The remaining animals were graded between 0 and 5 as per the established criteria. The total and graft corneal thickness and endothelial cell density progressively worsened with a higher grade of corneal opacity. The direct allosensitization of Th1 cells was significantly higher in mice with a higher grade of corneal opacity. At 16 weeks follow-up, the grafts remained stable with low opacity scores in syngeneic EK recipients; however, the opacity scores were higher and variable in allogeneic EK recipients. Conclusions: These findings establish a standardized protocol to assess the graft outcomes in a murine model of EK. Furthermore, we delineate the underlying immunological pathway that contributes to the immune-mediated rejection of grafts in this model.