Age-related T-cell cytokine profile parallels corneal disease severity in Sjogren's syndrome-like keratoconjunctivitis sicca in CD25KO mice

Abstract

Objectives: IL-2ralpha (CD25)(-/-) mice develop autoimmunity and lymphoproliferative disorders, including SS-like disease. The objective of this study was to evaluate the severity of corneal epithelial disease and T-cell cytokine profile in the ocular surface tissues of CD25KO mice.

Methods: CD25KO mice were evaluated at 8, 12 and 16 weeks of age. Corneal epithelial smoothness and corneal permeability were measured. Phenotype of infiltrating lymphocytes was evaluated by immunohistochemistry. Th-1, -2 and -17 associated factors were measured by real-time PCR in cornea and conjunctiva and by Luminex immunobead assay in tears.

Results: Compared with 8-week-old wild-type (WT) mice, CD25KO mice of the same age had significantly greater corneal irregularity and a significant increase in the number of CD4(+) and CD8(+) T cells infiltrating the conjunctiva. CD25KO mice had significantly higher levels of IL-6, TGF-beta1, IL-23R, IL-17A, IL-17F, IL-21, CCL20, IL-10, GATA-3 and IFN-gamma mRNA transcripts in their cornea and conjunctiva than WT mice at 8 weeks. IL-17A and IL-17F mRNA transcripts peaked at 12 weeks, whereas IFN-gamma spiked at 16 weeks in CD25KO mice. Increased expression of IL-17A and IL-17F at 12 weeks in CD25KO mice was accompanied by a worsening of corneal surface parameters and an increase of CD4(+) T cell infiltrating the cornea.

Conclusions: Disruption of IL-2 signalling in CD25KO mice results in age-dependent SS-like autoimmune lacrimal-keratoconjunctivitis. A mix of Th-1 and Th-17 cytokines was detected. The peak severity of corneal epithelial disease corresponded to the peak of IL-17 expression.

Fig. 1

Representative images of haemotoxylin and eosin-stained LG and SMG sections of aged C57BL/6 WT and CD25KO mice (from 8 to 16 weeks). Normal LG morphology was noted in the C57BL/6 WT at all time points, in contrast to CD25KO LG, which showed lymphocytic infiltration (circumscribed by black dotted lines) at all time points. The last images on the right are higher magnification of area demarcated by white dotted square in LG of both strains. In the CD25KO SMG sections, periductal infiltration was noted as early as 8 weeks (black dotted lines) and acinar atrophy was present at 16 weeks of age.

Fig. 2

Ocular surface evaluation of aged C57BL/6 and CD25KO mice from 8 to 16 weeks of age: corneal smoothness (A), corneal permeability to OGD (B) and conjunctival CD4⁺ T-cell immunostaining (in red, C). Merged images of laser-scanning IF confocal microscopy of cornea and conjunctiva sections stained for activated caspase 3 (in green, D) or processed for TUNEL assay (green, E) with propidium iodide nuclear counterstaining (in red). Orange colour in TUNEL indicates co-localization of green and red nuclear staining. Mean (s.d.) of corneal smoothness score (F), OGD staining score (G) and CD4⁺ T-cell density in the conjunctival epithelium (H). IL-17/IFN-γ mRNA ratio in corneal epithelial mRNA transcripts. Conj: conjunctiva; 8W: 8 weeks; 12W: 12 weeks; 16W: 16 weeks. *P < 0.05 vs C57BL/6 or CD25KO at 8 weeks of age; **P < 0.001 vs C57BL/6 or CD25KO at 8 weeks of age.

Fig. 3

Cytokine concentrations measured in tear fluid of C57BL/6 WT and CD25KO mice at different ages, using Luminex multiplex immunobead assay. Data are mean (s.d.) (error bars) for three separate experiments. Dotted lines indicate the lowest value in the linear portion of the curve, generated from the observed mean fluorescent intensities vs the observed concentrations of standards. *P < 0.05 when compared with C57BL/6 at 8 weeks of age.

Fig. 4

mRNA transcript levels of Th-17 inducers (A), Th-17 (B), Th-1 and Th-2 (C) pathways in aged conjunctiva of C57BL/6 and CD25KO mice (8, 12 and 16 weeks of age). Data are shown as the mean (s.e.m.) (n = 4/time point, *P < 0.05, **P < 0.01, ***P < 0.001 vs 8 W within the same strain, compared with 8 weeks; 8W: 8 weeks; 12W: 12 weeks; 16W: 16 weeks; ND: non-detectable.

Fig. 5

mRNA transcript levels in corneal epithelia of Th-17 inducers (A), Th-17 (B), Th-1 and Th-2 (C) pathways in aged C57BL/6 and CD25KO mice (8, 12 and 16 weeks of age). Data are shown as the mean (s.e.m.) (n = 4/time point, *P < 0.05, **P < 0.01, ***P < 0.001 vs 8W within the same strain, compared with 8 weeks; 8W: 8 weeks; 12W: 12 weeks; 16W: 16 weeks; ND: non-detectable.