Disruption of TGF-β signaling improves ocular surface epithelial disease in experimental autoimmune keratoconjunctivitis sicca

Abstract

Background: TGF-β is a pleiotropic cytokine that can have pro- or anti-inflammatory effects depending on the context. Elevated levels of bioactive TGF-β1 in tears and elevated TGF-β1mRNA transcripts in conjunctiva and minor salivary glands of human Sjögren's Syndrome patients has also been reported. The purpose of this study was to evaluate the response to desiccating stress (DS), an experimental model of dry eye, in dominant-negative TGF-β type II receptor (CD4-DNTGFβRII) mice. These mice have a truncated TGF-β receptor in CD4(+) T cells, rendering them unresponsive to TGF-β.

Methodology/principal findings: DS was induced by subcutaneous injection of scopolamine and exposure to a drafty low humidity environment in CD4-DNTGFβRII and wild-type (WT) mice, aged 14 weeks, for 5 days. Nonstressed (NS) mice served as controls. Parameters of ocular surface disease included corneal smoothness, corneal barrier function and conjunctival goblet cell density. NS CD4-DNTGFβRII at 14 weeks of age mice exhibited a spontaneous dry eye phenotype; however, DS improved their corneal barrier function and corneal surface irregularity, increased their number of PAS+ GC, and lowered CD4(+) T cell infiltration in conjunctiva. In contrast to WT, CD4-DNTGFβRII mice did not generate a Th-17 and Th-1 response, and they failed to upregulate MMP-9, IL-23, IL-17A, RORγT, IFN-γ and T-bet mRNA transcripts in conjunctiva. RAG1KO recipients of adoptively transferred CD4+T cells isolated from DS5 CD4-DNTGFβRII showed milder dry eye phenotype and less conjunctival inflammation than recipients of WT control.

Conclusions/significance: Our results showed that disruption of TGF-β signaling in CD4(+) T cells causes paradoxical improvement of dry eye disease in mice subjected to desiccating stress.

Figure 1. Age-related changes in wild-type (WT) and DNTGFBRII mice, from 8 to 14 weeks of age (8W and 14W, respectively).

A. Representative images of cornea sections immunostained for CD4 (in red) used to generate CD4 counts in B, in corneal epithelium (EPI) and corneal stroma (ST) in wild-type and DNTGFβRII mice at 8 and 14 weeks of age (8W,14W). Bar charts show mean ± SD of two independent experiment (final n = 5 for each group). C. Representative images of OGD corneal staining used to generate OGD intensity score in D. Bar charts show mean ± SD of three independent experiments (final n = 12 for each group). E. Corneal smoothness score. Bar charts show mean ± SD of three independent experiments (final n = 13 for each group). F. Representative images of conjunctiva sections immunostained for CD4 (in red) used to generate CD4 counts in G. Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). H. PAS+ conjunctival goblet cells counts. Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). I–K. Relative fold of expression in cornea (I), conjunctiva, (CJ, in J) and lacrimal gland (LG, in K) using the WT-8W as the calibrator. Bar charts show mean ± SD of three independent experiment (final n = 8 for each group). In graphs B, D,E, G, H **P<0.01;*** P<0.001 indicate within strain comparison In I–K graphs,*** P<0.001 indicates interstrain comparison.

Figure 2. Desiccating stress-induced changes in wild-type (WT) and DNTGFβRII mice at 14 weeks of age (14W).

Desiccating stress was induced for 5 days in both strains (DS5); nonstressed mice served as controls (NS). A. Representative images of OGD corneal staining used to generate OGD intensity score in B. Bar charts show mean ± SD of three independent experiments (final n = 12 for each group). C. CD4 counts in corneal epithelium (EPI) and corneal stroma (ST). Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). D. Representative images of conjunctiva sections immunostained for CD4 (in red) used to generate CD4 counts in E, in conjunctiva epithelium Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). F. PAS+ conjunctival goblet cells counts. Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). G–I. Relative fold of expression in cornea (I), conjunctiva, (CJ, in J) and lacrimal gland (LG, in K) using the WT-NS of each strain as the strain-calibrator. Bar charts show mean ± SD of three independent experiments (final n = 8 for each group). **P<0.01;*** P<0.001 indicate within WT comparison (NS vs. DS5) ∧ P<0.05;∧∧∧P<0.01 indicates DNTGFBRII comparison (NS vs. DS5).

Figure 3. CD4+ T cell proliferation.

Proliferation of CD4⁺ T cell in CD4⁺ T cells co-cultured in the presence of cornea and conjunctival tissues (CN and CJ, respectively) before (nonstressed [NS]) or after desiccating stress for 5 days [DS5] from wild-type (WT) and CD4-DNTGFβRII mice (DN). Bar charts show mean ± SD of two independent experiments (final n = 5 for each group).

Figure 4. Expression of chemokine receptors and adoptive transfer results in RAG1KO mice.

A–B Flow cytometry analysis of freshly isolated cells from ocular surface (OS, in A) and cervical lymph nodes (CLN, in B) from wild-type (WT) and DNTGFβRII mice before (nonstressed, NS) and after 5 days of desiccating stress (DS5).*P<0.05, within strain comparison. Bar charts show mean ± SD of three independent experiments (final n = 4 for each group for OS, final n = 6 for each group for CLN). C.Conjunctival sections stained for CD4⁺ T cells (in red) of RAG1KO recipient mice that received CD4+ cells isolated from WT and DNTGFβRII DS5 mice. Note CD4+T cell infiltration in goblet cell rich area of conjunctiva in WT-DS5 recipient mice. The black dotted circle is a higher magnification of area demarcated by blue dotted circle. Original magnification: 20×. Scale bar = 50 µm. D and E. Conjunctival CD4+T cell (in D) and PAS+ conjunctival goblet cells (in E) counts in RAG1KO recipient mice that received cells from CD4+ cells isolated from WT and DNTGFβRII mice after 5 days of desiccating stress (DS5). Bar charts show mean ± SD of three independent experiments (final n = 5 for each group) F.Relative change of IL-17A, IFN-γ and IL-13 mRNA transcripts in conjunctiva of RAG recipients. Asterisks indicate comparison to strain-specific controls. Bar charts show mean ± SD of two independent experiments (final n = 6 for each group).