NK Cells Promote Th-17 Mediated Corneal Barrier Disruption in Dry Eye

The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL).Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3(+)CD103(+) (~22%), CD3(+)γδ(+) (~9.6%), CD3(+)NK(+) (2%), CD3(-)NK(+) (~4.4%), CD3(+)CD8α (~0.9%), and CD4 (~0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17(+) cells in the cervical lymph nodes and generation of less pathogenic CD4(+)T cells. B6.nude recipient mice of adoptively transferred CD4(+)T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4(+)T cells.Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response

Disruption of TGF-β Signaling Improves Ocular Surface Epithelial Disease in Experimental Autoimmune Keratoconjunctivitis Sicca

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.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.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

Chemokine receptors CCR6 and CXCR3 are necessary for CD4(+) T cell mediated ocular surface disease in experimental dry eye disease.

CD4(+) T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4(+) T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4(+) T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4(+) T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease

Inflammatory cytokines are not produced in response to DS in the cornea and conjunctiva of CCR6KO and CXCR3KO CD4⁺ T cell recipient mice.

<p>Wild-type (WT), CCR6KO, or CXCR3KO CD4⁺ T cells from NS or DS5 mice were adoptively transferred to T cell deficient RAG1KO mice. mRNA analysis for inflammatory cytokines and matrix metalloproteinases (MMP) was performed on the corneal (A) and conjunctival (B) tissues of CD4⁺ T cell RAG1KO recipient mice. Mean±SD of transcript levels of corneal and conjunctival tissues. Results represent the mean±SD expression of 2-3 animals per strain/time point per experiment in two independent experiments for a total of 5-6 mice. *, P<0.05, **, P<0.001, ***, P<0.0001.</p

CCR6KO and CXCR3KO mice are resistant to dry eye disease.

<p><b>A.</b> Corneal permeability - Mean±SD of Oregon green dextran (OGD) permeability into the corneal epithelium of wild-type (WT), CCR6KO and CXCR3KO mice without (NS) or with 5 days of desiccating stress (DS5). OGD permeability was measured in five to seven mice per strain/time point/experiment. The graph represents the combined results of two independent experiments with a total of 10-14 mice per experimental group. <b>B.</b> Representative pictures of OGD staining of each strain. <b>C.</b> CD4⁺ T cell density – Mean±SD of the number of CD4⁺ cells per 100 µm of conjunctival epithelium of WT, CCR6KO, and CXCR3KO mice without (NS) or with 5 (DS5) or 10 (DS10) days of desiccating stress. <b>D.</b> Representative pictures of CD4⁺ T cell infiltration in each strain. * - indicates goblet cells; ↓ - indicates CD4⁺ T cells; CJ – conjunctiva; K – cornea. <b>E.</b> Goblet cell density - Mean±SD of the number of goblet cells per mm of conjunctival epithelium of WT, CCR6KO and CXCR3KO mice without (NS) or with 5 (DS5) or 10 (DS10) days of desiccating stress. Three sections from three animals were examined (N = 9) for each time point/mouse strain for both CD4⁺ T cell and goblet cell density. <b>F.</b> Representative pictures of PAS staining of each strain.</p

Th1 and Th17 associated cytokines are produced in the cervical lymph node but not the ocular surface.

<p><b>A.</b> Gene expression of IFN-γ, IL-17A, and IL-13 in the superficial cervical lymph node (CLN) of wild-type C57BL/6, CCR6KO, and CXCR3KO exposed to DS for 5 days (DS5) or not exposed (NS). <b>B.</b> Gene expression of IFN-γ, IL-17A, and IL-13 the ocular surface of wild-type C57BL/6, CCR6KO, and CXCR3KO exposed to DS for 5 days (DS5) or not exposed (NS). Results represent the mean±SD expression 2-3 animals per strain per time point in two independent experiments with a total of five mice. *, P<0.05, **, P<0.01.</p

Increased expression of CCR6⁺ and CXCR3⁺ cells in the cervical lymph node and at the ocular surface in response the desiccating stress.

<p><b>A.</b> The percentage of CCR6⁺CD4⁺ T cells in the superficial cervical lymph node (CLN) in mice exposed to no stress (NS) or five (DS5) days of desiccating stress (DS) was determined by flow cytometry. The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>B.</b> The percentage of CCR6⁺CD4⁺ T cells in the ocular surface (OS). The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>C.</b> The percentage of CXCR3⁺CD4⁺ T cells at the CLN. The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>D.</b> The percentage of CXCR3⁺CD4⁺ T cells at the OS. The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>E.</b> A representative dot plot of the percentage of CD4⁺ cells that are CCR6⁺ or CXCR3⁺ in the CLN.</p

Location of intra-epithelial lymphocytes in conjunctiva before and after desiccating stress.

<p>Representative digital pictures of immunohistochemical staining of CD4⁺, CD8α⁺, CD103⁺, γδTCR⁺, NK⁺ in the conjunctivae of nonstressed (NS) C57BL/6 mice and after desiccating stress for 5 or 10 days (DS5, DS10). Inset in γδTCR row shows γδTCR in skin, which was used as positive control. Original magnification 40X; scale bar 20 µm. Number insets represent cell counts in the goblet cell rich of the conjunctiva in immunostained tissue sections in conjunctival epithelium of C57BL/6 mice. Data represents mean ± SD of cells/mm. Experiments were repeated three times with two mice per group per experiment. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.01 comparison vs. NS control.</p

Adoptive transfer results.

<p><b>A</b> Mean± SD of IL-17 ELISPOTs showing IL-17- producing cells isolated from the ocular surface (OS) and CD4⁺ T cells isolated from spleen and cervical lymph nodes (CLN) in donor mice that received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) antibody before (non-stressed, NS) and after 5 days of desiccating stress (DS5). Experiments were repeated two times with at least five mice per group per experiment. <b>B</b> Representative images of OGD corneal staining used to generate OGD intensity score in <b>C.</b> Bar charts show mean ± SD of three independent experiments with five mice for each group per experiment. <b>D-G-</b> Laser scanning immunofluorescent confocal microscopy of cornea immunostained for MMP-3 (in <b>D</b>) and MMP-9 (in <b>F</b>) in nude mice that received CD4⁺T cells isolated from donor mice treated with systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Bar graphs are mean±SD of fluorescence intensity measured in corneal epithelium for MMP-3 (E) and MMP-9 (G) of a total of two independent experiments with at least three mice per group per experiment. <b>H-K</b>-Gene expression analyses showing mean± SD (copies) of IL-17A (in <b>H</b>), CCL20 (in <b>I</b>), matrix metalloproteinases (MMP)-3 (in <b>J</b>) and MMP-9 (in <b>K</b>) mRNA transcripts in cornea epithelia of nude mice that received CD4⁺T cells isolated from donor mice that had received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Data represents mean ± SD. Experiments were repeated two times with at least three mice per group per experiment.</p