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NK cells promote Th-17 mediated corneal barrier disruption in dry eye - PubMed

NK cells promote Th-17 mediated corneal barrier disruption in dry eye

Xiaobo Zhang et al. PLoS One. 2012.

Abstract

Background: The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL).

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and have the following conflicts: M. Stern, C. Schaumburg and K. Siemasko are employees of Allergan Inc. M. Stern also holds a visiting professorship appointment at Baylor College of Medicine. However, Allergan Inc. was not a funder in this project. Their presence as authors does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Location of intra-epithelial lymphocytes in conjunctiva before and after desiccating stress.

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.

Figure 2
Figure 2. Charcterization of intraepithelial lymphocytes (IELs) in the normal murine conjunctiva.

Representative flow cytometry analysis of cells isolated from ocular surface (A) or spleen (B) stained with CD3 antibody and γδ (GDTCR), CD8α, NK1.1 and CD103 markers. (+) = positive cells, (–) =  negative cells. Lymphocytes were gated based on characteristic light-scatter properties (“gated cells”, circled population on far left panels), subsequently gated based on forward scatter height vs. forward scatter area (FSC-A) and propridium iodide live/dead exclusion (“live cells”, not shown). Numbers in the quadrants indicate the percentage of cells of one representative experiment. SSC-A = side scatter area.

Figure 3
Figure 3. Cytokine burst from NK positive populations after DS.

mRNA levels in NK/NKT positive (+) and NK/NKT negative (–) cells isolated from nonstressed (NS) spleen and ocular surface (OS) and at different time points after desiccating stress (DS; DS1 = DS for 1 day, DS5 = DS for 5 days, DS10 = DS10 for 10 days). Unfractionated spleen was used as calibrator. Experiments were repeated two times with at least three samples per group per experiment. Because the standard deviation is relatively small compared to the levels of IL-6, IL-23 and IL-17A expression, the error bars do not show in the graph. * indicates p<0.05, *** indicates p<0.001 comparison vs. NS control NK/NKT+. ∧ indicates P<0.05, ∧∧∧ indicates p<0.001 comparison vs. NS control NK/NKT−.

Figure 4
Figure 4. Flow cytometry analysis of dendritic cell maturation. A&B

Flow cytometry analysis of CD11c+ and dual CD11c+MHC II+ cells (A) CD11b+ and dual CD11b+MHC II+ cells (B) in the CLN in non-stressed controls (NS) and after desiccating stress for (DS) for 1 (DS1) or 5 (DS5). A separate group of mice received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) antibody after 5 days of desiccating stress (DS5). Data represents mean± SD. Experiments were repeated three times with at least four mice per group per experiment. *indicates p<0.05 comparison; ** indicates p<0.01 comparison, *** indicates p<0.001 comparison.

Figure 5
Figure 5. Adoptive transfer results.

A 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 Representative images of OGD corneal staining used to generate OGD intensity score in C. Bar charts show mean ± SD of three independent experiments with five mice for each group per experiment. D-G- Laser scanning immunofluorescent confocal microscopy of cornea immunostained for MMP-3 (in D) and MMP-9 (in F) 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. H-K-Gene expression analyses showing mean± SD (copies) of IL-17A (in H), CCL20 (in I), matrix metalloproteinases (MMP)-3 (in J) and MMP-9 (in K) 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.

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